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IκBα deficiency in brain leads to elevated basal neuroinflammation and attenuated response following traumatic brain injury: implications for functional recovery.

Lian H, Shim DJ, Gaddam SS, Rodriguez-Rivera J, Bitner BR, Pautler RG, Robertson CS, Zheng H - Mol Neurodegener (2012)

Bottom Line: By generating mice with brain-specific deletion of IκBα, we show that IκBα deficiency does not compromise normal brain development.However, basal neuroinflammation detected by GFAP and Iba1 immunoreactivity is elevated.We conclude that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Huffington Center on Aging Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT

Background: The transcription factor NFκB is an important mediator of cell survival and inflammation in the immune system. In the central nervous system (CNS), NFκB signaling has been implicated in regulating neuronal survival following acute pathologic damage such as traumatic brain injury (TBI) and stroke. NFκB is normally bound by the principal inhibitory protein, IκBα, and sequestered in the cytoplasm. Activation of NFκB requires the degradation of IκBα, thereby freeing NFκB to translocate to the nucleus and activate the target genes. Mice deficient in IκBα display deregulated and sustained NFκB activation and early postnatal lethality, highlighting a critical role of IκBα in NFκB regulation.

Results: We investigated the role of IκBα in regulating NFκB activity in the brain and the effects of the NFκB/IκBα pathway in mediating neuroinflammation under both physiological and brain injury conditions. We report that astrocytes, but not neurons, exhibit prominent NFκB activity, and that basal NFκB activity in astrocytes is elevated in the absence of IκBα. By generating mice with brain-specific deletion of IκBα, we show that IκBα deficiency does not compromise normal brain development. However, basal neuroinflammation detected by GFAP and Iba1 immunoreactivity is elevated. This leads to impaired inflammatory responses following TBI and worsened brain damage including higher blood brain barrier permeability, increased injury volumes and enlarged ventricle volumes.

Conclusions: We conclude that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation. We further demonstrate that IκBα plays an important role in regulating NFκB activity in the brain and a robust NFκB/IκBα-mediated neuroinflammatory response immediately following TBI is beneficial.

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IκBα deletion results in increased NFκB activity in astrocytes. (A and B) Representative images of wild-type (WT) and IκBα knockout (KO) primary neurons, either under basal condition (A) or treated with 50 ng/ml TNFα for 30 min (B), followed by staining against neuronal marker Neuronal nuclei (NeuN) and NFκB subunit p65, and displayed as individual or merged images. Note the uniform p65 staining in NeuN-positive neurons under both conditions and the cells that undergo p65 nuclear translocation in response to TNFα (B) are NeuN-negative. (C and D) Representative images of primary astrocytes, either under basal condition (C) or treated with TNFα (D), followed by staining against astroglia marker GFAP and p65, and displayed as individual or merged images. Note clear p65 nuclear translocation in GFAP-positive astrocytes. (E) Representative p65 immunostaining of primary WT and KO astroglia cultures. (F) Quantification of relative p65 nuclear to cytoplasmic intensity in WT and IκBα KO neurons with or without TNFα. (G) Quantification of relative p65 nuclear versus cytoplasmic fluorescence intensity in WT and IκBα KO astrocytes in the presence or absence of TNFα stimulation, documenting increased basal p65 in IκBα KO sample and greatly enhanced nuclear p65 upon TNFα treatment in both WT and KO cultures. N = 7-10/genotype. (H) p65 ELISA quantification of nuclear preparations from adult (2–3 month) Ctrl and IκBα astroglia-specific knockout (GcKO) hippocampal samples. N = 3/genotype. ns: non-significant; *p < 0.05. Scale bar: 25 μm.
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Figure 2: IκBα deletion results in increased NFκB activity in astrocytes. (A and B) Representative images of wild-type (WT) and IκBα knockout (KO) primary neurons, either under basal condition (A) or treated with 50 ng/ml TNFα for 30 min (B), followed by staining against neuronal marker Neuronal nuclei (NeuN) and NFκB subunit p65, and displayed as individual or merged images. Note the uniform p65 staining in NeuN-positive neurons under both conditions and the cells that undergo p65 nuclear translocation in response to TNFα (B) are NeuN-negative. (C and D) Representative images of primary astrocytes, either under basal condition (C) or treated with TNFα (D), followed by staining against astroglia marker GFAP and p65, and displayed as individual or merged images. Note clear p65 nuclear translocation in GFAP-positive astrocytes. (E) Representative p65 immunostaining of primary WT and KO astroglia cultures. (F) Quantification of relative p65 nuclear to cytoplasmic intensity in WT and IκBα KO neurons with or without TNFα. (G) Quantification of relative p65 nuclear versus cytoplasmic fluorescence intensity in WT and IκBα KO astrocytes in the presence or absence of TNFα stimulation, documenting increased basal p65 in IκBα KO sample and greatly enhanced nuclear p65 upon TNFα treatment in both WT and KO cultures. N = 7-10/genotype. (H) p65 ELISA quantification of nuclear preparations from adult (2–3 month) Ctrl and IκBα astroglia-specific knockout (GcKO) hippocampal samples. N = 3/genotype. ns: non-significant; *p < 0.05. Scale bar: 25 μm.

Mentions: As a predominant inhibitor and downstream target of NFκB, IκBα plays an essential role in regulating NFκB activity. Both activation and suppression of NFκB by IκBα have been reported, likely mediated in a cell type-specific manner[19,20,26]. Accordingly, we prepared primary neuronal and primary astrocyte cultures from the germline IκBα knockout neonates and performed immunostaining using an antibody that recognizes the major NFκB subunit, p65 (Figure 2). Neurons from wild-type (WT) and IκBα knockout (KO) mice showed similar weak and homogeneous p65 distribution over the whole cell body (Figure 2A), indicative of non-specific staining. This is corroborated by the similar staining pattern following treatment with the potent NFκB activator TNFα (Figure 2, B and quantified in F). Of interest, the cells that show clear p65 nuclear translocation in response to TNFα are NeuN-negative, likely due to the contamination of glial cells in the culture (Figure 2B). In contrast, strong cytoplasmic and weak nuclear staining can be detected in GFAP-positive astrocytes using the anti-p65 antibody (Figure 2C). The specificity of the signal was further confirmed by the robust nuclear translocation of p65 upon treating the cultures with TNFα (Figure 2D). Measurement of nuclear and cytoplasmic fluorescence intensity from the control and IκBα KO astrocytes under basal condition revealed higher nuclear to cytoplasmic ratio (Nuc/Cyt) in the absence of IκBα (Figure 2G, WT vs. KO), suggesting that deleting IκBα leads to higher basal NFκB activity. TNFα treatment led to strong nuclear translocation of TNFα. However, p65 Nuc/Cyt ratio after TNFα treatment showed similar values between WT and KO astrocytes likely caused by oversaturated nuclear signal intensity (Figure 2G, WT+ TNFα vs. KO + TNFα). The enhanced basal nuclear NFκB in KO astrocytes is further strengthened by quantifying the nuclear NFκB levels in IκBα conditional knockout mice in which IκBα is specifically deleted in astrocytes by crossing the IκBα floxed allele with the GFAP-Cre transgenic mice[28] (Figure 2H). Overall, our results provide strong support for the notion that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation and that loss of IκBα in astrocytes results in higher basal NFκB activity. Nevertheless, we cannot exclude the possibility that neuronal NFkB may be induced under pathological conditions in vivo.


IκBα deficiency in brain leads to elevated basal neuroinflammation and attenuated response following traumatic brain injury: implications for functional recovery.

Lian H, Shim DJ, Gaddam SS, Rodriguez-Rivera J, Bitner BR, Pautler RG, Robertson CS, Zheng H - Mol Neurodegener (2012)

IκBα deletion results in increased NFκB activity in astrocytes. (A and B) Representative images of wild-type (WT) and IκBα knockout (KO) primary neurons, either under basal condition (A) or treated with 50 ng/ml TNFα for 30 min (B), followed by staining against neuronal marker Neuronal nuclei (NeuN) and NFκB subunit p65, and displayed as individual or merged images. Note the uniform p65 staining in NeuN-positive neurons under both conditions and the cells that undergo p65 nuclear translocation in response to TNFα (B) are NeuN-negative. (C and D) Representative images of primary astrocytes, either under basal condition (C) or treated with TNFα (D), followed by staining against astroglia marker GFAP and p65, and displayed as individual or merged images. Note clear p65 nuclear translocation in GFAP-positive astrocytes. (E) Representative p65 immunostaining of primary WT and KO astroglia cultures. (F) Quantification of relative p65 nuclear to cytoplasmic intensity in WT and IκBα KO neurons with or without TNFα. (G) Quantification of relative p65 nuclear versus cytoplasmic fluorescence intensity in WT and IκBα KO astrocytes in the presence or absence of TNFα stimulation, documenting increased basal p65 in IκBα KO sample and greatly enhanced nuclear p65 upon TNFα treatment in both WT and KO cultures. N = 7-10/genotype. (H) p65 ELISA quantification of nuclear preparations from adult (2–3 month) Ctrl and IκBα astroglia-specific knockout (GcKO) hippocampal samples. N = 3/genotype. ns: non-significant; *p < 0.05. Scale bar: 25 μm.
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Figure 2: IκBα deletion results in increased NFκB activity in astrocytes. (A and B) Representative images of wild-type (WT) and IκBα knockout (KO) primary neurons, either under basal condition (A) or treated with 50 ng/ml TNFα for 30 min (B), followed by staining against neuronal marker Neuronal nuclei (NeuN) and NFκB subunit p65, and displayed as individual or merged images. Note the uniform p65 staining in NeuN-positive neurons under both conditions and the cells that undergo p65 nuclear translocation in response to TNFα (B) are NeuN-negative. (C and D) Representative images of primary astrocytes, either under basal condition (C) or treated with TNFα (D), followed by staining against astroglia marker GFAP and p65, and displayed as individual or merged images. Note clear p65 nuclear translocation in GFAP-positive astrocytes. (E) Representative p65 immunostaining of primary WT and KO astroglia cultures. (F) Quantification of relative p65 nuclear to cytoplasmic intensity in WT and IκBα KO neurons with or without TNFα. (G) Quantification of relative p65 nuclear versus cytoplasmic fluorescence intensity in WT and IκBα KO astrocytes in the presence or absence of TNFα stimulation, documenting increased basal p65 in IκBα KO sample and greatly enhanced nuclear p65 upon TNFα treatment in both WT and KO cultures. N = 7-10/genotype. (H) p65 ELISA quantification of nuclear preparations from adult (2–3 month) Ctrl and IκBα astroglia-specific knockout (GcKO) hippocampal samples. N = 3/genotype. ns: non-significant; *p < 0.05. Scale bar: 25 μm.
Mentions: As a predominant inhibitor and downstream target of NFκB, IκBα plays an essential role in regulating NFκB activity. Both activation and suppression of NFκB by IκBα have been reported, likely mediated in a cell type-specific manner[19,20,26]. Accordingly, we prepared primary neuronal and primary astrocyte cultures from the germline IκBα knockout neonates and performed immunostaining using an antibody that recognizes the major NFκB subunit, p65 (Figure 2). Neurons from wild-type (WT) and IκBα knockout (KO) mice showed similar weak and homogeneous p65 distribution over the whole cell body (Figure 2A), indicative of non-specific staining. This is corroborated by the similar staining pattern following treatment with the potent NFκB activator TNFα (Figure 2, B and quantified in F). Of interest, the cells that show clear p65 nuclear translocation in response to TNFα are NeuN-negative, likely due to the contamination of glial cells in the culture (Figure 2B). In contrast, strong cytoplasmic and weak nuclear staining can be detected in GFAP-positive astrocytes using the anti-p65 antibody (Figure 2C). The specificity of the signal was further confirmed by the robust nuclear translocation of p65 upon treating the cultures with TNFα (Figure 2D). Measurement of nuclear and cytoplasmic fluorescence intensity from the control and IκBα KO astrocytes under basal condition revealed higher nuclear to cytoplasmic ratio (Nuc/Cyt) in the absence of IκBα (Figure 2G, WT vs. KO), suggesting that deleting IκBα leads to higher basal NFκB activity. TNFα treatment led to strong nuclear translocation of TNFα. However, p65 Nuc/Cyt ratio after TNFα treatment showed similar values between WT and KO astrocytes likely caused by oversaturated nuclear signal intensity (Figure 2G, WT+ TNFα vs. KO + TNFα). The enhanced basal nuclear NFκB in KO astrocytes is further strengthened by quantifying the nuclear NFκB levels in IκBα conditional knockout mice in which IκBα is specifically deleted in astrocytes by crossing the IκBα floxed allele with the GFAP-Cre transgenic mice[28] (Figure 2H). Overall, our results provide strong support for the notion that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation and that loss of IκBα in astrocytes results in higher basal NFκB activity. Nevertheless, we cannot exclude the possibility that neuronal NFkB may be induced under pathological conditions in vivo.

Bottom Line: By generating mice with brain-specific deletion of IκBα, we show that IκBα deficiency does not compromise normal brain development.However, basal neuroinflammation detected by GFAP and Iba1 immunoreactivity is elevated.We conclude that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Huffington Center on Aging Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT

Background: The transcription factor NFκB is an important mediator of cell survival and inflammation in the immune system. In the central nervous system (CNS), NFκB signaling has been implicated in regulating neuronal survival following acute pathologic damage such as traumatic brain injury (TBI) and stroke. NFκB is normally bound by the principal inhibitory protein, IκBα, and sequestered in the cytoplasm. Activation of NFκB requires the degradation of IκBα, thereby freeing NFκB to translocate to the nucleus and activate the target genes. Mice deficient in IκBα display deregulated and sustained NFκB activation and early postnatal lethality, highlighting a critical role of IκBα in NFκB regulation.

Results: We investigated the role of IκBα in regulating NFκB activity in the brain and the effects of the NFκB/IκBα pathway in mediating neuroinflammation under both physiological and brain injury conditions. We report that astrocytes, but not neurons, exhibit prominent NFκB activity, and that basal NFκB activity in astrocytes is elevated in the absence of IκBα. By generating mice with brain-specific deletion of IκBα, we show that IκBα deficiency does not compromise normal brain development. However, basal neuroinflammation detected by GFAP and Iba1 immunoreactivity is elevated. This leads to impaired inflammatory responses following TBI and worsened brain damage including higher blood brain barrier permeability, increased injury volumes and enlarged ventricle volumes.

Conclusions: We conclude that, in the CNS, astrocyte is the primary cell type subject to NFκB regulation. We further demonstrate that IκBα plays an important role in regulating NFκB activity in the brain and a robust NFκB/IκBα-mediated neuroinflammatory response immediately following TBI is beneficial.

Show MeSH
Related in: MedlinePlus