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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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Generation and characterization of brain IκBα conditional knockout (cKO) mice. (A) Breeding scheme used for generating the IκBα cKO mice and the littermate controls (Ctrl). (B) Real-time quantitative PCR (qPCR) analysis of IκBα mRNA (N = 3/genotype) and (C) Western blot analysis of IκBα protein levels in brain lysates of 2–3 month old adult cKO and Ctrl hippocampal tissue. (D) Representative images of Nissl-stained brain sections of 10–12 month old Ctrl and cKO mice. Scale bar: 1 mm. White boxes highlight the sampling region for counting pyramidal neuronal number. (E) Quantification of pyramidal neurons in hippocampal CA1 region (N = 5/genotype) and (F) brain weight measurements of Ctrl and IκBα cKO mice (N = 9-14/genotype).
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Figure 1: Generation and characterization of brain IκBα conditional knockout (cKO) mice. (A) Breeding scheme used for generating the IκBα cKO mice and the littermate controls (Ctrl). (B) Real-time quantitative PCR (qPCR) analysis of IκBα mRNA (N = 3/genotype) and (C) Western blot analysis of IκBα protein levels in brain lysates of 2–3 month old adult cKO and Ctrl hippocampal tissue. (D) Representative images of Nissl-stained brain sections of 10–12 month old Ctrl and cKO mice. Scale bar: 1 mm. White boxes highlight the sampling region for counting pyramidal neuronal number. (E) Quantification of pyramidal neurons in hippocampal CA1 region (N = 5/genotype) and (F) brain weight measurements of Ctrl and IκBα cKO mice (N = 9-14/genotype).

Mentions: We generated mice with specific deletion of IκBα in the brain (IκBα cKO) by crossing an IκBα floxed allele[26] with the Nestin-Cre[27] transgenic mice (Figure 1A). Littermate IκBαfl/- mice without Cre transgene was used as controls (Ctrl). Quantitative real-time PCR (qPCR) analysis showed that the IκBα mRNA level was significantly reduced (Figure 1B). Immunoblotting of IκBα revealed a similar level of reduction in protein expression in the cKO mice (Figure 1C). In contrast to the IκBα germline knockout mice, which are early postnatal lethal, the IκBα cKO mice are viable and overtly normal. Nissl staining of adult IκBα cKO brains revealed indistinguishable morphology compared to the littermate controls (Figure 1D). Further quantification of pyramidal neurons in CA1 region showed no overt neurodegeneration in the IκBα cKO mice (Figure 1E) and this is in agreement with their normal brain weight (Figure 1F).


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)

Generation and characterization of brain IκBα conditional knockout (cKO) mice. (A) Breeding scheme used for generating the IκBα cKO mice and the littermate controls (Ctrl). (B) Real-time quantitative PCR (qPCR) analysis of IκBα mRNA (N = 3/genotype) and (C) Western blot analysis of IκBα protein levels in brain lysates of 2–3 month old adult cKO and Ctrl hippocampal tissue. (D) Representative images of Nissl-stained brain sections of 10–12 month old Ctrl and cKO mice. Scale bar: 1 mm. White boxes highlight the sampling region for counting pyramidal neuronal number. (E) Quantification of pyramidal neurons in hippocampal CA1 region (N = 5/genotype) and (F) brain weight measurements of Ctrl and IκBα cKO mice (N = 9-14/genotype).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Generation and characterization of brain IκBα conditional knockout (cKO) mice. (A) Breeding scheme used for generating the IκBα cKO mice and the littermate controls (Ctrl). (B) Real-time quantitative PCR (qPCR) analysis of IκBα mRNA (N = 3/genotype) and (C) Western blot analysis of IκBα protein levels in brain lysates of 2–3 month old adult cKO and Ctrl hippocampal tissue. (D) Representative images of Nissl-stained brain sections of 10–12 month old Ctrl and cKO mice. Scale bar: 1 mm. White boxes highlight the sampling region for counting pyramidal neuronal number. (E) Quantification of pyramidal neurons in hippocampal CA1 region (N = 5/genotype) and (F) brain weight measurements of Ctrl and IκBα cKO mice (N = 9-14/genotype).
Mentions: We generated mice with specific deletion of IκBα in the brain (IκBα cKO) by crossing an IκBα floxed allele[26] with the Nestin-Cre[27] transgenic mice (Figure 1A). Littermate IκBαfl/- mice without Cre transgene was used as controls (Ctrl). Quantitative real-time PCR (qPCR) analysis showed that the IκBα mRNA level was significantly reduced (Figure 1B). Immunoblotting of IκBα revealed a similar level of reduction in protein expression in the cKO mice (Figure 1C). In contrast to the IκBα germline knockout mice, which are early postnatal lethal, the IκBα cKO mice are viable and overtly normal. Nissl staining of adult IκBα cKO brains revealed indistinguishable morphology compared to the littermate controls (Figure 1D). Further quantification of pyramidal neurons in CA1 region showed no overt neurodegeneration in the IκBα cKO mice (Figure 1E) and this is in agreement with their normal brain weight (Figure 1F).

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