IkappaB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-kappaB in the central nervous system - PubMed (original) (raw)

. 2011 Apr;134(Pt 4):1184-98.

doi: 10.1093/brain/awq359. Epub 2011 Feb 10.

Nicolas Zeller, Geert van Loo, Doron Merkler, Alexander Mildner, Daniel Erny, Klaus-Peter Knobeloch, John R Bethea, Ari Waisman, Markus Knust, Domenico Del Turco, Thomas Deller, Thomas Blank, Josef Priller, Wolfgang Brück, Manolis Pasparakis, Marco Prinz

Affiliations

IkappaB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-kappaB in the central nervous system

Jenni Raasch et al. Brain. 2011 Apr.

Abstract

The IκB kinase complex induces nuclear factor kappa B activation and has recently been recognized as a key player of autoimmunity in the central nervous system. Notably, IκB kinase/nuclear factor kappa B signalling regulates peripheral myelin formation by Schwann cells, however, its role in myelin formation in the central nervous system during health and disease is largely unknown. Surprisingly, we found that brain-specific IκB kinase 2 expression is dispensable for proper myelin assembly and repair in the central nervous system, but instead plays a fundamental role for the loss of myelin in the cuprizone model. During toxic demyelination, inhibition of nuclear factor kappa B activation by conditional ablation of IκB kinase 2 resulted in strong preservation of central nervous system myelin, reduced expression of proinflammatory mediators and a significantly attenuated glial response. Importantly, IκB kinase 2 depletion in astrocytes, but not in oligodendrocytes, was sufficient to protect mice from myelin loss. Our results reveal a crucial role of glial cell-specific IκB kinase 2/nuclear factor kappa B signalling for oligodendrocyte damage during toxic demyelination. Thus, therapies targeting IκB kinase 2 function in non-neuronal cells may represent a promising strategy for the treatment of distinct demyelinating central nervous system diseases.

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Figures

Figure 1

Figure 1

The canonical NF-κB pathway activator IKK2 is dispensable for formation of myelin in the CNS. (A) Immunoblot analysis of lysates demonstrating brain and neuroectoderm-specific deletion of IKK2. Data are representative of four independent experiments. Lysates from total brain, cortex and subcortex were generated from mice at the age of 6–8 weeks. Lysates from astrocytes and microglia derived from newborn mice primary cell cultures. (B) Proteolipid protein expression reveals proper myelination and the absence of any gross abnormalities within the CNS in the absence of IKK2 (left panel) at the age of 6–8 weeks. GFAP-expressing astrocytes are normally distributed within the white matter tract of the corpus callosum (right). Analysis was performed with mice at the age of 6–8 weeks. Scale bars = 500 µm (left panel) and 50 µm (right panel). (C) Ultrastructural micrographs showing myelination in the corpus callosum at different time points of myelination. Scale bar = 400 nm (left). Measurements of axon diameter, myelin thickness, G ratio and percentage of myelin fibres in the corpus callosum of three and 25-week old IKK2CNS-KO (filled squares) or wild-type (WT) mice (open squares, right). There are no differences between the mean group values for any parameter in either genotype or at either developmental stage. Each point represents the mean value from at least 100 individual axonal measurements. (D) The number of oligodendroglial progenitor cells and mature oligodendrocytes was quantitatively examined on histological sections of corpus callosum and cortex in 2–3-week old IKK2CNS-KO and IKK2CNS-wild-type mice. Olig2 immunohistochemistry (left) and quantification thereof (right). Data are expressed as mean ± SEM of at least three mice per group. (E) Unaltered oligodendrocyte differentiation and morphology in IKK2CNS-KO mice. Primary oligodendrocytes were cultured and kept for different maturation stages. Double immunofluorescence with DAPI for nuclear staining (blue) and NG2 (red), myelin basic protein (red) or CNP (green), respectively (left panel). Scale bar = 10 µm. Quantification of differentially expressed maturation marker for oligodendrocytes after three days in culture (right). Data indicate the mean of cells expressing the differentiation marker. Data are representative of two independent experiments. MBP = myelin basic protein.

Figure 2

Figure 2

Inhibition of NF-κB signalling in the brain ameliorates demyelination and reduces induction of toxic cytokines in the CNS. (A) NF-κB p65 translocation is induced in neuroectodermal and myeloid cells of the brain during acute demyelination. Immunofluorescence of p65 depicts strong nuclear signal in numerous GFAP+ astrocytes (arrow, left panel), Nogo-A+ oligodendrocytes (arrows, middle panel) and MAC-3+ microglia (arrows, right panel). Inserts illustrate the absence of nuclear p65 translocation in neuroectodermal glia cells of IKK2CNS-KO mice. Scale bar = 25 µm. (B) Histopathological analysis of the corpus callosum from IKK2CNS-wild-type and IKK2CNS-KO mice 5 weeks after cuprizone treatment. Sections were examined for demyelination by luxol fast blue (LFB), for microglia by MAC-3 and for astrogliosis by GFAP immunohistochemistry. Representative sections are shown left and quantification thereof is depicted on the right. (C) The presence of NG2+ cells in the corpus callosum of mice treated with cuprizone is shown by immunofluorescence (red). Nuclei are stained in blue (DAPI). Inserts depict higher magnification. Scale bars = 30 µm and 7 µm (inserts). The number of NG2+ cells per mm2 is shown on the right (mean ± SEM). (D) Quantification of cytokine and chemokine messenger RNA expression in the corpus callosum of IKK2CNS-wild-type and IKK2CNS-KO mice 5 weeks after cuprizone treatment. (E) Electron micrographs depicting highly preserved myelin sheaths in mice lacking IKK2 in all neuroectodermal cells of the brain. The number of myelinated axons is greatly reduced in the presence of canonical NF-κB signalling as quantified in at least 100 axons of a diameter ≥250 nm after acute corpus callosum demyelination. Scale bar = 400 nm. *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 3

Figure 3

Gene expression pattern is temporally altered during chronic demyelination in the absence of IKK2. (A) Time course of demyelination (upper panel) and the number Nogo-A+ oligodendrocytes (lower panel) in IKK2CNS-wild-type (open squares) and IKK2CNS-KO (filled squares) mice. Data show mean ± SEM from at least three mice per group. (B) Levels of myelin transcripts in the corpus callosum of IKK2CNS-wild-type (open squares) and IKK2CNS-KO (filled squares) mice treated with cuprizone over time, measured by quantitative real-time polymerase chain reaction, normalized to GAPDH and expressed in relation to untreated mice (n ≥ 3). Mean ± SEM from at least three mice per group is depicted. (C) Attenuated demyelination is found in the corpus callosum of chronically treated animals after 10 weeks in the absence of canonical NF-κB activation. Representative histological sections are shown left and the quantification is given right. Data show mean ± SEM from one representative experiment out of two with at least five mice per group. *P < 0.05. Scale bar = 300 µm. LFB = luxol fast blue; MAG (myelin associated glycoprotein) = MAC-3 for microglia and GFAP for astrocytes.

Figure 4

Figure 4

NF-κB signalling in mature oligodendrocytes is not pathogenic during demyelination. (A) Analysis of paraffin sections indicate the same pattern (left) and extend (right) of demyelination (luxol fast blue, LFB), infiltrating microglia (MAC-3) as well as astrogliosis (GFAP) after cuprizone treatment for 5 weeks. Data show mean ± SEM from one representative experiment out of three. Scale bar = 300 µm. (B) Similar number of NG2+ progenitor cells in IKK2Oligo-wild-type and IKK2Oligo-KO mice 5 weeks after cuprizone treatment. Representative mean ± SEM of two similar experiments is shown. Scale bar = 20 µm.

Figure 5

Figure 5

Diverse lesion pattern and different inflammatory make up in two distinct demyelination models. (A) Immunohistochemical examination of inflammation and myelin and axonal damage during acute toxic demyelination in the lysolecithin model at peak of disease (4 days after injection) compared with the maximal response after cuprizone challenge (5 weeks of treatment). MAC-3 staining revealed a plethora of macrophages/microglia in both lesion models, whereas significantly more GFAP+ astrocytes were found in cuprizone-induced lesions during acute myelin loss. Lymphocytes (CD3 for T cells, B220 for B cells) were scarce in both models. Notably, amyloid precursor protein (APP) deposits representing axonal damage were clearly detectable only on the lysolecithin model. (B) Histopathology of infiltration and axonal damage during remyelination after lysolecithin (14 days after injection) and the cuprizone (5 weeks treatment and 5 weeks recovery) model. Data show mean ± SEM from one representative experiment out of two with at least four or five mice per group. Scale bar = 400 μm low magnification; scale bar = 40 μm high magnification. *P < 0.05, **P < 0.01, LFB-periodic acid-Schiff = luxol fast blue-periodic acid Schiff.

Figure 6

Figure 6

Remyelination in the CNS is independent from canonical NF-κB activation by IKK2. (A) Transmission electron microscopy revealed a similar extent of demyelination induced by lysolecithin four days after treatment (left). Percentage of myelinated fibres is shown for IKK2CNS-wild-type (open squares) and IKK2CNS-KO mice (filled squares). Each symbol represents the mean value from at least 20 individual measurements in each mouse. (B) Morphologically compatible structures of myelin in the remyelination phase 14 days after lysolecithin exposure in both genotypes (left). Measurements of the percentages of myelinated axons, myelin thickness, G ratio and axon diameter in the spinal cord of IKK2CNS-wild-type (open squares) and IKK2CNS-KO mice (filled squares). Scale bar = 400 nm. Each point represents the mean value from at least 50 individual axonal measurements.

Figure 7

Figure 7

Selective downregulation of NF-κB activation in neuroectodermal significantly inhibits myelin loss. (A) Laser microdissection of GFAP+ astrocytes (upper row) and CD11b+ microglia (lower row) from the lesion site in the corpus callosum after 5 weeks of cuprizone treatment. Location of cells before (left) and after (right) microdissection. Scale bar = 50 µm. (B) Quantification of cytokine and chemokine messenger RNA in microdissected astrocytes and microglia reveals IKK2-dependent suppression of gene induction in astrocytes. Messenger RNA levels were normalized to β-actin and compared with untreated controls. Mean ± SEM from at least three mice per group are exhibited. *P < 0.05. (C) Histopathological analysis of myelin loss and accompanying gliosis in GFAP-IκBα-dn and wild-type (WT) mice 5 weeks after cuprizone treatment. Sections were assessed for myelin damage by luxol fast blue (LFB) and for gliosis (immunostaining for astrocytes by GFAP and for microglia by MAC-3, respectively). Neuropathological changes were quantified (below) and statistically significant changes were marked with *P < 0.05. (D) Expression of demyelination-associated genes in the corpus callosum of GFAP-IκBα-dn (black bars) and wild-type (white bars) animals during toxic demyelination. Data are expressed as mean ± SEM of at least four animals per group and significant changes are indicated *P < 0.05.

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