Limits...
Borna disease virus phosphoprotein impairs the developmental program controlling neurogenesis and reduces human GABAergic neurogenesis.

Scordel C, Huttin A, Cochet-Bernoin M, Szelechowski M, Poulet A, Richardson J, Benchoua A, Gonzalez-Dunia D, Eloit M, Coulpier M - PLoS Pathog. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1161, Maisons-Alfort, France; ANSES, UMR Virologie, Maisons-Alfort, France; Université Paris-Est, Ecole Nationale Vétérinaire d'Alfort, UMR Virologie, Maisons-Alfort, France.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

bdv-p expression does not alter neuronal specification but induces a reduction in the GABAergic subpopulation.Transduced hNPCs expressing bdv-p and their matched NT controls were induced to differentiate for 0, 7, 10, 14, 21 and 28 days and immunostained with antibodies directed against markers of different stages of differentiation. (A) immunostaining of hNPCs differentiated for 28 days with an anti-Sox2 antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Time-course analyses showing the percentage of (B) Sox2-positive cells and (C) HuC/D-positive cells. (D) Immunostaining of hNPCs differentiated for 14 days with antibodies against HuC/D and GABA. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (E) Time-course analysis showing the percentage of huC/D- and GABA-positive cells in the total neuronal population. Results are representative of 2 (B) and 3 (C and E) independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.005. nd, non-determined.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4414417&req=5

ppat.1004859.g005: bdv-p expression does not alter neuronal specification but induces a reduction in the GABAergic subpopulation.Transduced hNPCs expressing bdv-p and their matched NT controls were induced to differentiate for 0, 7, 10, 14, 21 and 28 days and immunostained with antibodies directed against markers of different stages of differentiation. (A) immunostaining of hNPCs differentiated for 28 days with an anti-Sox2 antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Time-course analyses showing the percentage of (B) Sox2-positive cells and (C) HuC/D-positive cells. (D) Immunostaining of hNPCs differentiated for 14 days with antibodies against HuC/D and GABA. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (E) Time-course analysis showing the percentage of huC/D- and GABA-positive cells in the total neuronal population. Results are representative of 2 (B) and 3 (C and E) independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.005. nd, non-determined.

Mentions: To define the stage at which neuronal differentiation was impaired with greater precision, we performed a time-course study in which the number of Sox2- and HuC/D-positive cells was monitored throughout differentiation. Sox2 is a universal marker of neural progenitors that is known to be down-regulated during differentiation when progenitors become post-mitotic [36]. We reasoned that if a pool of bdv-p-expressing hNPCs were blocked at the progenitor stage, Sox2-positive cells would be more numerous than in control cells. We thus labeled bdv-p-expressing hNPCs and their NT matched controls with an antibody directed against Sox2 from 0 to 28 days of differentiation. As expected, at the progenitor stage (day 0), 100% of NT hNPCs were Sox-2 positive and their number continuously decreased during differentiation (Fig 5A and 5B). They represented approximately 60% of the population at day 14 and 40% at day 28. It was somewhat surprising to observe that as many as 60% of hNPCs still expressed Sox2 after 14 days of differentiation, since at that time approximately 90% of the cells had already differentiated into either βIII-Tubulin- or GFAP-positive cells. This indicated that loss of Sox2 expression is gradual during the differentiation process. Most notably, no difference was observed in the percentage of Sox2-positive cells between bdv-p- and NT- cells at any time point studied, indicating that P does not prevent the cells from exiting the progenitor stage. Next, to address whether P blocks cell entry into the neuronal pathway, bdv-p-expressing hNPCs and their NT matched controls were labeled with an antibody directed against HuC/D, a nuclear neuronal marker that is expressed as soon as the neuroblasts exit the proliferation cell cycle [37]. In NT cells, approximately 60% of cells were HuC/D-positive at day 7 and their number rose up to day 10, at which time it remained constant up until day 28 (Fig 5C). This showed that by day 10, commitment to the neuronal lineage has been completed. The estimate of the neuronal population based on HuC/D immunostaining at 14 days of differentiation in NT cells was somewhat higher (approximately 80%) than that previously determined on the basis of βIII-Tubulin immunostaining (approximately 60%, Fig 3). This is possibly due to variability between experiments and/or to differences in the manner in which cells were enumerated (automatically for HuC/D and manually for βIII-Tubulin). To address this issue, HuC/D- and βIII-Tubulin-positive cells were both manually enumerated in one single experiment, at day 14 of differentiation. Similar values were obtained (approximately 70%), indicating that, at that time, all NT cells that had entered a neuronal pathway had acquired the βIII-Tubulin marker. Most notably, at every time point studied, no significant difference in the percentage of HuC/D-positive cells was observed between bdv-p-expressing cells and their NT controls (Fig 5C), indicating that P does not impair neuronal commitment. Thus, altogether, our results suggest that P impairs the acquisition of a mature neuronal phenotype.


Borna disease virus phosphoprotein impairs the developmental program controlling neurogenesis and reduces human GABAergic neurogenesis.

Scordel C, Huttin A, Cochet-Bernoin M, Szelechowski M, Poulet A, Richardson J, Benchoua A, Gonzalez-Dunia D, Eloit M, Coulpier M - PLoS Pathog. (2015)

bdv-p expression does not alter neuronal specification but induces a reduction in the GABAergic subpopulation.Transduced hNPCs expressing bdv-p and their matched NT controls were induced to differentiate for 0, 7, 10, 14, 21 and 28 days and immunostained with antibodies directed against markers of different stages of differentiation. (A) immunostaining of hNPCs differentiated for 28 days with an anti-Sox2 antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Time-course analyses showing the percentage of (B) Sox2-positive cells and (C) HuC/D-positive cells. (D) Immunostaining of hNPCs differentiated for 14 days with antibodies against HuC/D and GABA. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (E) Time-course analysis showing the percentage of huC/D- and GABA-positive cells in the total neuronal population. Results are representative of 2 (B) and 3 (C and E) independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.005. nd, non-determined.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414417&req=5

ppat.1004859.g005: bdv-p expression does not alter neuronal specification but induces a reduction in the GABAergic subpopulation.Transduced hNPCs expressing bdv-p and their matched NT controls were induced to differentiate for 0, 7, 10, 14, 21 and 28 days and immunostained with antibodies directed against markers of different stages of differentiation. (A) immunostaining of hNPCs differentiated for 28 days with an anti-Sox2 antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Time-course analyses showing the percentage of (B) Sox2-positive cells and (C) HuC/D-positive cells. (D) Immunostaining of hNPCs differentiated for 14 days with antibodies against HuC/D and GABA. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. (E) Time-course analysis showing the percentage of huC/D- and GABA-positive cells in the total neuronal population. Results are representative of 2 (B) and 3 (C and E) independent experiments performed in triplicate. Statistical analyses were performed using the Mann-Whitney test. ***, p < 0.005. nd, non-determined.
Mentions: To define the stage at which neuronal differentiation was impaired with greater precision, we performed a time-course study in which the number of Sox2- and HuC/D-positive cells was monitored throughout differentiation. Sox2 is a universal marker of neural progenitors that is known to be down-regulated during differentiation when progenitors become post-mitotic [36]. We reasoned that if a pool of bdv-p-expressing hNPCs were blocked at the progenitor stage, Sox2-positive cells would be more numerous than in control cells. We thus labeled bdv-p-expressing hNPCs and their NT matched controls with an antibody directed against Sox2 from 0 to 28 days of differentiation. As expected, at the progenitor stage (day 0), 100% of NT hNPCs were Sox-2 positive and their number continuously decreased during differentiation (Fig 5A and 5B). They represented approximately 60% of the population at day 14 and 40% at day 28. It was somewhat surprising to observe that as many as 60% of hNPCs still expressed Sox2 after 14 days of differentiation, since at that time approximately 90% of the cells had already differentiated into either βIII-Tubulin- or GFAP-positive cells. This indicated that loss of Sox2 expression is gradual during the differentiation process. Most notably, no difference was observed in the percentage of Sox2-positive cells between bdv-p- and NT- cells at any time point studied, indicating that P does not prevent the cells from exiting the progenitor stage. Next, to address whether P blocks cell entry into the neuronal pathway, bdv-p-expressing hNPCs and their NT matched controls were labeled with an antibody directed against HuC/D, a nuclear neuronal marker that is expressed as soon as the neuroblasts exit the proliferation cell cycle [37]. In NT cells, approximately 60% of cells were HuC/D-positive at day 7 and their number rose up to day 10, at which time it remained constant up until day 28 (Fig 5C). This showed that by day 10, commitment to the neuronal lineage has been completed. The estimate of the neuronal population based on HuC/D immunostaining at 14 days of differentiation in NT cells was somewhat higher (approximately 80%) than that previously determined on the basis of βIII-Tubulin immunostaining (approximately 60%, Fig 3). This is possibly due to variability between experiments and/or to differences in the manner in which cells were enumerated (automatically for HuC/D and manually for βIII-Tubulin). To address this issue, HuC/D- and βIII-Tubulin-positive cells were both manually enumerated in one single experiment, at day 14 of differentiation. Similar values were obtained (approximately 70%), indicating that, at that time, all NT cells that had entered a neuronal pathway had acquired the βIII-Tubulin marker. Most notably, at every time point studied, no significant difference in the percentage of HuC/D-positive cells was observed between bdv-p-expressing cells and their NT controls (Fig 5C), indicating that P does not impair neuronal commitment. Thus, altogether, our results suggest that P impairs the acquisition of a mature neuronal phenotype.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1161, Maisons-Alfort, France; ANSES, UMR Virologie, Maisons-Alfort, France; Université Paris-Est, Ecole Nationale Vétérinaire d'Alfort, UMR Virologie, Maisons-Alfort, France.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus