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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

Expression of bdv-p or bdv-x gene does not alter hNPCs at the undifferentiated stage.RNAs from bdv-p- and bdv-x-expressing hNPCs and their matched NT controls were analyzed by RT-qPCR for expression of (A) Nestin and (B) Sox2. Proliferation of bdv-p and bdv-x-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. ns, non-significant (p > 0.5).
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ppat.1004859.g002: Expression of bdv-p or bdv-x gene does not alter hNPCs at the undifferentiated stage.RNAs from bdv-p- and bdv-x-expressing hNPCs and their matched NT controls were analyzed by RT-qPCR for expression of (A) Nestin and (B) Sox2. Proliferation of bdv-p and bdv-x-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. ns, non-significant (p > 0.5).

Mentions: At the undifferentiated stage, transgenic hNPCs expressing either the gfp, bdv-p or bdv-x gene were morphologically indistinguishable from their non-transduced (NT) matched controls (S1 Fig). Transgene expression had no impact on cell survival, as observed by light microscopy examination (S1 Fig), and the presence of the viral proteins, P or X, did not modify the expression of Nestin and Sox2, two markers of the undifferentiated stage (Fig 2A and 2B). We next examined whether the viral genes influenced the proliferative capacity of hNPCs. Transgene-expressing hNPCs and their NT matched controls were cultured in the presence of growth factors—EGF and bFGF—and proliferation was determined by evaluation of both BrdU incorporation and mitochondrial dehydrogenase activity. No significant difference was observed between bdv-p- and bdv-x-expressing hNPCs and their matched NT controls (Fig 2C, 2D and 2E). Since, however, an effect on proliferation may have been masked by the presence of growth factors, we measured proliferation after growth factor withdrawal by enumerating hNPCs at day 0 and day 4 of differentiation using DAPI staining. At day 4, NT hNPCs were 5 to 7.5 fold more numerous than at day 0, showing that cells continued to proliferate in the first days of differentiation. No difference, however, was observed in bdv-p- and bdv-x-expressing hNPCs compared with their matched NT controls (Fig 2F and 2G), demonstrating that transgenic hNPCs were not impaired in their capacity to proliferate. Thus, the viral P and X proteins did not alter hNPCs at the undifferentiated stage.


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)

Expression of bdv-p or bdv-x gene does not alter hNPCs at the undifferentiated stage.RNAs from bdv-p- and bdv-x-expressing hNPCs and their matched NT controls were analyzed by RT-qPCR for expression of (A) Nestin and (B) Sox2. Proliferation of bdv-p and bdv-x-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. ns, non-significant (p > 0.5).
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Related In: Results  -  Collection

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

ppat.1004859.g002: Expression of bdv-p or bdv-x gene does not alter hNPCs at the undifferentiated stage.RNAs from bdv-p- and bdv-x-expressing hNPCs and their matched NT controls were analyzed by RT-qPCR for expression of (A) Nestin and (B) Sox2. Proliferation of bdv-p and bdv-x-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. ns, non-significant (p > 0.5).
Mentions: At the undifferentiated stage, transgenic hNPCs expressing either the gfp, bdv-p or bdv-x gene were morphologically indistinguishable from their non-transduced (NT) matched controls (S1 Fig). Transgene expression had no impact on cell survival, as observed by light microscopy examination (S1 Fig), and the presence of the viral proteins, P or X, did not modify the expression of Nestin and Sox2, two markers of the undifferentiated stage (Fig 2A and 2B). We next examined whether the viral genes influenced the proliferative capacity of hNPCs. Transgene-expressing hNPCs and their NT matched controls were cultured in the presence of growth factors—EGF and bFGF—and proliferation was determined by evaluation of both BrdU incorporation and mitochondrial dehydrogenase activity. No significant difference was observed between bdv-p- and bdv-x-expressing hNPCs and their matched NT controls (Fig 2C, 2D and 2E). Since, however, an effect on proliferation may have been masked by the presence of growth factors, we measured proliferation after growth factor withdrawal by enumerating hNPCs at day 0 and day 4 of differentiation using DAPI staining. At day 4, NT hNPCs were 5 to 7.5 fold more numerous than at day 0, showing that cells continued to proliferate in the first days of differentiation. No difference, however, was observed in bdv-p- and bdv-x-expressing hNPCs compared with their matched NT controls (Fig 2F and 2G), demonstrating that transgenic hNPCs were not impaired in their capacity to proliferate. Thus, the viral P and X proteins did not alter hNPCs at the undifferentiated stage.

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