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A role for the canonical nuclear factor-κB pathway in coupling neurotrophin-induced differential survival of developing spiral ganglion neurons.

Vandenbosch R, Chocholova E, Robe PA, Wang Y, Lambert C, Moonen G, Lallemend F, Malgrange B, Hadjab S - Front Cell Neurosci (2013)

Bottom Line: Using dissociated cultures of embryonic and postnatal spiral ganglion neurons, we observed a specific requirement of NFκB for BDNF but not NT3-dependent neuronal survival during a particular postnatal time window that corresponds to a period of neuronal cell death and hair cell innervation refinement in the developing cochlea.Consistently, postnatal p65 knockout mice showed a specific decreased number in type II spiral ganglion neurons.Taken together, these results identify NFκB as a type II neuron-specific factor that participates in the selective survival effects of BDNF and NT3 signaling on developing spiral ganglion neurons.

View Article: PubMed Central - PubMed

Affiliation: Groupe Interdisciplinaire de Génoprotéomique Appliquée-Neurosciences, Developmental Neurobiology Unit, University of Liège Liège, Belgium.

ABSTRACT
Neurotrophins are key players of neural development by controlling cell death programs. However, the signaling pathways that mediate their selective responses in different populations of neurons remain unclear. In the mammalian cochlea, sensory neurons differentiate perinatally into type I and II populations both expressing TrkB and TrkC, which bind respectively brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). How these two neuronal populations respond differentially to these two neurotrophins remains unknown. Here, we report in rat the segregation of the nuclear factor-κB (NFκB) subunit p65 specifically within the type II population postnatally. Using dissociated cultures of embryonic and postnatal spiral ganglion neurons, we observed a specific requirement of NFκB for BDNF but not NT3-dependent neuronal survival during a particular postnatal time window that corresponds to a period of neuronal cell death and hair cell innervation refinement in the developing cochlea. Consistently, postnatal p65 knockout mice showed a specific decreased number in type II spiral ganglion neurons. Taken together, these results identify NFκB as a type II neuron-specific factor that participates in the selective survival effects of BDNF and NT3 signaling on developing spiral ganglion neurons.

No MeSH data available.


Distribution of p65 and p50 proteins in the developing cochea.(A–L) Representative confocal images for the expression of p65 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (A–C, E–G, I–K; scale bar: 100 μm) and corresponding organs of Corti (OC; D,H,L; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in A (A’), represents immunoblotting for p65 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. (M–X) Representative confocal images for the expression of p50 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (M–O, Q–R, U–W; scale bar: 100 μm) and OC (P,T,X; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in M (M’), represents immunoblotting for p50 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. On the same immunoblot, the specific band corresponding to p105 is not shown.
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Figure 2: Distribution of p65 and p50 proteins in the developing cochea.(A–L) Representative confocal images for the expression of p65 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (A–C, E–G, I–K; scale bar: 100 μm) and corresponding organs of Corti (OC; D,H,L; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in A (A’), represents immunoblotting for p65 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. (M–X) Representative confocal images for the expression of p50 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (M–O, Q–R, U–W; scale bar: 100 μm) and OC (P,T,X; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in M (M’), represents immunoblotting for p50 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. On the same immunoblot, the specific band corresponding to p105 is not shown.

Mentions: The marked decrease of p65 expression in whole SG observed during the first postnatal week may indicate a general downregulation of p65 or a selective expression within a specific neuronal population. To address this issue, we examined the temporal and spatial distribution of p65 expression on cochlear sections of different developmental stages using antibodies against p65, βIII-tubulin (a general neuronal marker; Fanarraga et al., 1999) and peripherin, a type III intermediate filament protein that serves as a reliable marker for type II neurons at postnatal and adult stages (Hafidi, 1998; Lallemend et al., 2007). At late embryonic stage, we observed that p65 is widely expressed in the whole SG, staining most if not all sensory neurons (Figures 2A–C). In contrast, after birth, p65 immunostaining could be visualized in only a few neurons (Figures 2E–G). This pattern of expression was maintained at adult stage, where the small size of the p65 immunopositive neurons indicates that they are type II neurons (Figures 2I–K). A close analysis of the immunostaining at the level of the organ of Corti revealed a clear staining below the OHCs, but only during the early postnatal period (Figures 2D,H,L). A further analysis of both peripherin and p65 expression confirmed that these p65 positive neurons were type II neurons (Figure 3A). OHCs innervation consists of type II afferents from the SG and efferents that convey information from the brain to the cochlea (Knipper et al., 1996; Wiechers et al., 1999). To distinguish between these two types of fibers and confirm that the p65 positive projections below the OHCs are only type II afferents, we labeled postnatal cochlea sections for both p65 and synaptophysin, a specific maker for presynaptic terminations that stain efferents below the hair cells (Knipper et al., 1995). Synaptophysin positive nerve endings were seen below each OHC, but never colocalized with the p65 positive projections, indicating that these last projections are exclusively type II afferents (Figure 3B). Later during development, around P11 when hearing starts in rat, the expression of p65 could no longer be seen within the SG (data not shown). It reappeared however, at around P15-16 in type II neuronal cell bodies, but not in their projections, an expression profile that was maintained at adult stage (Figure 2L).


A role for the canonical nuclear factor-κB pathway in coupling neurotrophin-induced differential survival of developing spiral ganglion neurons.

Vandenbosch R, Chocholova E, Robe PA, Wang Y, Lambert C, Moonen G, Lallemend F, Malgrange B, Hadjab S - Front Cell Neurosci (2013)

Distribution of p65 and p50 proteins in the developing cochea.(A–L) Representative confocal images for the expression of p65 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (A–C, E–G, I–K; scale bar: 100 μm) and corresponding organs of Corti (OC; D,H,L; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in A (A’), represents immunoblotting for p65 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. (M–X) Representative confocal images for the expression of p50 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (M–O, Q–R, U–W; scale bar: 100 μm) and OC (P,T,X; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in M (M’), represents immunoblotting for p50 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. On the same immunoblot, the specific band corresponding to p105 is not shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Distribution of p65 and p50 proteins in the developing cochea.(A–L) Representative confocal images for the expression of p65 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (A–C, E–G, I–K; scale bar: 100 μm) and corresponding organs of Corti (OC; D,H,L; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in A (A’), represents immunoblotting for p65 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. (M–X) Representative confocal images for the expression of p50 subunit (green) and the neuronal marker TUJ1 (red) on rat SG (M–O, Q–R, U–W; scale bar: 100 μm) and OC (P,T,X; scale bar: 40 μm) from E19, P5 and P35 rat cochlear sections. Inset in M (M’), represents immunoblotting for p50 from P5 rat SG total protein extract which reveals a specific single band staining at the expected molecular weight. On the same immunoblot, the specific band corresponding to p105 is not shown.
Mentions: The marked decrease of p65 expression in whole SG observed during the first postnatal week may indicate a general downregulation of p65 or a selective expression within a specific neuronal population. To address this issue, we examined the temporal and spatial distribution of p65 expression on cochlear sections of different developmental stages using antibodies against p65, βIII-tubulin (a general neuronal marker; Fanarraga et al., 1999) and peripherin, a type III intermediate filament protein that serves as a reliable marker for type II neurons at postnatal and adult stages (Hafidi, 1998; Lallemend et al., 2007). At late embryonic stage, we observed that p65 is widely expressed in the whole SG, staining most if not all sensory neurons (Figures 2A–C). In contrast, after birth, p65 immunostaining could be visualized in only a few neurons (Figures 2E–G). This pattern of expression was maintained at adult stage, where the small size of the p65 immunopositive neurons indicates that they are type II neurons (Figures 2I–K). A close analysis of the immunostaining at the level of the organ of Corti revealed a clear staining below the OHCs, but only during the early postnatal period (Figures 2D,H,L). A further analysis of both peripherin and p65 expression confirmed that these p65 positive neurons were type II neurons (Figure 3A). OHCs innervation consists of type II afferents from the SG and efferents that convey information from the brain to the cochlea (Knipper et al., 1996; Wiechers et al., 1999). To distinguish between these two types of fibers and confirm that the p65 positive projections below the OHCs are only type II afferents, we labeled postnatal cochlea sections for both p65 and synaptophysin, a specific maker for presynaptic terminations that stain efferents below the hair cells (Knipper et al., 1995). Synaptophysin positive nerve endings were seen below each OHC, but never colocalized with the p65 positive projections, indicating that these last projections are exclusively type II afferents (Figure 3B). Later during development, around P11 when hearing starts in rat, the expression of p65 could no longer be seen within the SG (data not shown). It reappeared however, at around P15-16 in type II neuronal cell bodies, but not in their projections, an expression profile that was maintained at adult stage (Figure 2L).

Bottom Line: Using dissociated cultures of embryonic and postnatal spiral ganglion neurons, we observed a specific requirement of NFκB for BDNF but not NT3-dependent neuronal survival during a particular postnatal time window that corresponds to a period of neuronal cell death and hair cell innervation refinement in the developing cochlea.Consistently, postnatal p65 knockout mice showed a specific decreased number in type II spiral ganglion neurons.Taken together, these results identify NFκB as a type II neuron-specific factor that participates in the selective survival effects of BDNF and NT3 signaling on developing spiral ganglion neurons.

View Article: PubMed Central - PubMed

Affiliation: Groupe Interdisciplinaire de Génoprotéomique Appliquée-Neurosciences, Developmental Neurobiology Unit, University of Liège Liège, Belgium.

ABSTRACT
Neurotrophins are key players of neural development by controlling cell death programs. However, the signaling pathways that mediate their selective responses in different populations of neurons remain unclear. In the mammalian cochlea, sensory neurons differentiate perinatally into type I and II populations both expressing TrkB and TrkC, which bind respectively brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). How these two neuronal populations respond differentially to these two neurotrophins remains unknown. Here, we report in rat the segregation of the nuclear factor-κB (NFκB) subunit p65 specifically within the type II population postnatally. Using dissociated cultures of embryonic and postnatal spiral ganglion neurons, we observed a specific requirement of NFκB for BDNF but not NT3-dependent neuronal survival during a particular postnatal time window that corresponds to a period of neuronal cell death and hair cell innervation refinement in the developing cochlea. Consistently, postnatal p65 knockout mice showed a specific decreased number in type II spiral ganglion neurons. Taken together, these results identify NFκB as a type II neuron-specific factor that participates in the selective survival effects of BDNF and NT3 signaling on developing spiral ganglion neurons.

No MeSH data available.