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The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

Larocque D, Fragoso G, Huang J, Mushynski WE, Loignon M, Richard S, Almazan G - PLoS ONE (2009)

Bottom Line: In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation.Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, Department of Oncology and Medicine, McGill University, Montréal, Québec, Canada.

ABSTRACT

Background: The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.

Methodology/principal findings: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.

Conclusions/significance: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

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The QKI isoforms induce Schwann cell cycle arrest.Primary Schwann cells/neuron co-cultures were infected for 48 hr with control GFP adenovirus (A and B) and adenoviruses encoding for QKI-6 (C and D), QKI-7 (E and F), and the combination of QKI-6/7 (G and H). The green fluorescence denotes infected cells (panels A, C, E and G). Incorporation of BrdU (16 hr pulse) was detected with an anti-BrdU antibody followed by a goat anti-mouse conjugated to Alexa 546 (red) (B, D, F and H). Arrowheads denote the GFP positive Schwann cells that are also BrdU positive in the controls infected with GFP-adenovirus. (I). The expression of each QKI isoform causes inhibition of Schwann cell proliferation. The graph shows the percentage of GFP positive cells that are also BrdU positive. The proliferation was quantified from n>500 cells from three different experiments (p<0.001, ANOVA). The error bars represent +/− standard deviation of the mean.
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pone-0005867-g001: The QKI isoforms induce Schwann cell cycle arrest.Primary Schwann cells/neuron co-cultures were infected for 48 hr with control GFP adenovirus (A and B) and adenoviruses encoding for QKI-6 (C and D), QKI-7 (E and F), and the combination of QKI-6/7 (G and H). The green fluorescence denotes infected cells (panels A, C, E and G). Incorporation of BrdU (16 hr pulse) was detected with an anti-BrdU antibody followed by a goat anti-mouse conjugated to Alexa 546 (red) (B, D, F and H). Arrowheads denote the GFP positive Schwann cells that are also BrdU positive in the controls infected with GFP-adenovirus. (I). The expression of each QKI isoform causes inhibition of Schwann cell proliferation. The graph shows the percentage of GFP positive cells that are also BrdU positive. The proliferation was quantified from n>500 cells from three different experiments (p<0.001, ANOVA). The error bars represent +/− standard deviation of the mean.

Mentions: The quaking viable mice (qkv) exhibit PNS dysmyelination [27], [28], [29]. Moreover, the expression of the cytoplasmic QKI-6 and QKI-7 is lost in the Schwann cells of qkv mice. These observations suggest that QKI-6 and QKI-7 may regulate Schwann cell differentiation [7]. To investigate the functional significance of QKI in Schwann cells, primary rat co-cultures of Schwann cells and neurons obtained from the dorsal root ganglia (DRG), which reflect the in vivo context of glial cells in the proximity of neurons [32], were transduced with adenoviruses expressing QKI-6 or QKI-7. Each virus also encodes the green fluorescent protein (GFP) for the microscopic monitoring of transduction efficiency and ∼95% of the Schwann cells were transduced, as visualized by the presence of the green fluorescence (data not shown and Figure 1). The adenovirus transduction resulted in an ∼6- to 8-fold increase in QKI-6 and QKI-7 protein expression (Figure S1). This elevated level of QKI-6 and QKI-7 expression is similar to what is observed in mice brains during the time of myelination in the CNS (Figure S2). In these conditions, the proliferation of Schwann cells was inhibited by either QKI-6 or QKI-7 or both QKI-6/7 isoforms, as determined by immunofluorescence detection of bromodeoxyuridine (BrdU) incorporation (Figure 1A–H). A quantitative analysis from three separate experiments to determine the percentage of BrdU-positive Schwann cells set the rate of incorporation at ∼3% and ∼2% for cells transduced with QKI-6 or QKI-7 expressing adenoviruses, respectively, and to ∼20% for cells transduced with the control vector GFP expressing adenovirus (Figure 1I). These findings demonstrate that both QKI-6 and QKI-7 isoforms inhibit Schwann cell proliferation.


The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

Larocque D, Fragoso G, Huang J, Mushynski WE, Loignon M, Richard S, Almazan G - PLoS ONE (2009)

The QKI isoforms induce Schwann cell cycle arrest.Primary Schwann cells/neuron co-cultures were infected for 48 hr with control GFP adenovirus (A and B) and adenoviruses encoding for QKI-6 (C and D), QKI-7 (E and F), and the combination of QKI-6/7 (G and H). The green fluorescence denotes infected cells (panels A, C, E and G). Incorporation of BrdU (16 hr pulse) was detected with an anti-BrdU antibody followed by a goat anti-mouse conjugated to Alexa 546 (red) (B, D, F and H). Arrowheads denote the GFP positive Schwann cells that are also BrdU positive in the controls infected with GFP-adenovirus. (I). The expression of each QKI isoform causes inhibition of Schwann cell proliferation. The graph shows the percentage of GFP positive cells that are also BrdU positive. The proliferation was quantified from n>500 cells from three different experiments (p<0.001, ANOVA). The error bars represent +/− standard deviation of the mean.
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pone-0005867-g001: The QKI isoforms induce Schwann cell cycle arrest.Primary Schwann cells/neuron co-cultures were infected for 48 hr with control GFP adenovirus (A and B) and adenoviruses encoding for QKI-6 (C and D), QKI-7 (E and F), and the combination of QKI-6/7 (G and H). The green fluorescence denotes infected cells (panels A, C, E and G). Incorporation of BrdU (16 hr pulse) was detected with an anti-BrdU antibody followed by a goat anti-mouse conjugated to Alexa 546 (red) (B, D, F and H). Arrowheads denote the GFP positive Schwann cells that are also BrdU positive in the controls infected with GFP-adenovirus. (I). The expression of each QKI isoform causes inhibition of Schwann cell proliferation. The graph shows the percentage of GFP positive cells that are also BrdU positive. The proliferation was quantified from n>500 cells from three different experiments (p<0.001, ANOVA). The error bars represent +/− standard deviation of the mean.
Mentions: The quaking viable mice (qkv) exhibit PNS dysmyelination [27], [28], [29]. Moreover, the expression of the cytoplasmic QKI-6 and QKI-7 is lost in the Schwann cells of qkv mice. These observations suggest that QKI-6 and QKI-7 may regulate Schwann cell differentiation [7]. To investigate the functional significance of QKI in Schwann cells, primary rat co-cultures of Schwann cells and neurons obtained from the dorsal root ganglia (DRG), which reflect the in vivo context of glial cells in the proximity of neurons [32], were transduced with adenoviruses expressing QKI-6 or QKI-7. Each virus also encodes the green fluorescent protein (GFP) for the microscopic monitoring of transduction efficiency and ∼95% of the Schwann cells were transduced, as visualized by the presence of the green fluorescence (data not shown and Figure 1). The adenovirus transduction resulted in an ∼6- to 8-fold increase in QKI-6 and QKI-7 protein expression (Figure S1). This elevated level of QKI-6 and QKI-7 expression is similar to what is observed in mice brains during the time of myelination in the CNS (Figure S2). In these conditions, the proliferation of Schwann cells was inhibited by either QKI-6 or QKI-7 or both QKI-6/7 isoforms, as determined by immunofluorescence detection of bromodeoxyuridine (BrdU) incorporation (Figure 1A–H). A quantitative analysis from three separate experiments to determine the percentage of BrdU-positive Schwann cells set the rate of incorporation at ∼3% and ∼2% for cells transduced with QKI-6 or QKI-7 expressing adenoviruses, respectively, and to ∼20% for cells transduced with the control vector GFP expressing adenovirus (Figure 1I). These findings demonstrate that both QKI-6 and QKI-7 isoforms inhibit Schwann cell proliferation.

Bottom Line: In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation.Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, Department of Oncology and Medicine, McGill University, Montréal, Québec, Canada.

ABSTRACT

Background: The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.

Methodology/principal findings: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR.

Conclusions/significance: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

Show MeSH
Related in: MedlinePlus