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Short Communication |
B signalling
1 Institute of Neuropathology, University Hospital of Zürich, Zürich, Switzerland
2 Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, School of Medicine, University of California, San Diego, USA
3 Department of Neuropathology, University of Freiburg, Freiburg, Germany
4 Institute for Genetics, University of Cologne, Cologne, Germany
Correspondence
A. Aguzzi
adriano.aguzzi{at}usz.ch
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ABSTRACT |
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B (NF-B) activity in the brain parallels the first pathological changes. The NF-B pathway is essential for proliferation, regulation of apoptosis and immune responses involving induction of inflammation. The IB kinase (IKK) signalosome is crucial for NF-B signalling, consisting of the catalytic IKK
/IKKβ subunits and the regulatory IKK
subunit. This study investigated the impact of NF-B signalling on prion disease in mouse models with a central nervous system (CNS)-restricted elimination of IKKβ or IKK in nearly all neuroectodermal cells, including neurons, astrocytes and oligodendrocytes, and in mice containing a non-phosphorylatable IKK subunit (IKKAA/AA). In contrast to previously published data, the observed results showed no evidence supporting the hypothesis that impaired NF-B signalling in the CNS impacts on prion pathogenesis.
These authors contributed equally to this work. 
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MAIN TEXT |
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). The latter include Creutzfeldt–Jakob disease in humans, scrapie in sheep and goat, bovine spongiform encephalopathy in cattle, and chronic wasting disease in cervids (Aguzzi & Polymenidou, 2004). The causative agent of TSEs is the prion, which is thought to consist of an abnormally folded, aggregated isoform (PrPSc) of the host-encoded cellular prion protein (PrPC) (McKinley et al., 1983). The histopathological changes that accompany TSEs include neuronal death, spongiosis and pronounced activation of glia, invariably leading to death of the affected individuals (Aguzzi, 2006). Neuronal cell death has been proposed to occur via apoptotic mechanisms in prion disease (Cronier et al., 2004; Fuhrmann et al., 2007; Hetz et al., 2003). However, the precise molecular and biochemical mechanisms underpinning the subsequent neuropathology are not understood.
The nuclear factor-B (NF-B) pathway is involved in a variety of physiological and pathological responses, and plays an essential role in immune responses involving induction of inflammation, proliferation and the regulation of apoptosis (Bonizzi et al., 2004; Karin, 2006). In acute and chronic neurodegenerative conditions such as Alzheimer's disease, NF-B is activated in neurons and glia, and it has been proposed that NF-B modulates these processes (Bourteele et al., 2007). Both deleterious and beneficial roles of NF-B signalling are conceivable (Mattson, 2005). Activation of NF-B might promote survival of neurons by inducing the expression of genes that encode anti-apoptotic proteins. Alternatively, NF-B may contribute to neuronal degeneration through the production and release of inflammatory cytokines, reactive oxygen molecules, or excitotoxins in microglia and astrocytes. In prion disease, NF-B activity was shown to be enhanced in parallel with the first neuronal pathological changes (Kim et al., 1999). Furthermore, the cytotoxic synthetic peptide PrP106–126 activates NF-B in microglia in vitro (Bacot et al., 2003). Notably, it has been claimed that NF-B activity leads to mitochondrial apoptosis after prion infection (Bourteele et al., 2007). Despite these findings, it remains elusive whether NF-B activation is protective or pathogenic in prion disease. The IB kinase (IKK) signalosome, which consists of the IKK and IKKβ catalytic subunits and the IKK regulatory subunit, is an essential component of the NF-B pathway and is necessary for NF-B activation through pro-inflammatory signals (Ghosh & Karin, 2002). The regulatory subunit IKK was first identified as an NF-B essential modulator and is therefore also named NEMO (Yamaoka et al., 1998).
As constitutive inactivation of the NF-B pathway leads to embryonic lethality, we opted to study the impact of the canonical (classical) NF-B pathway in prion disease in recently developed tissue-specific knockout models (van Loo et al., 2006). Here, central nervous system (CNS)-restricted elimination of the IKKβ or IKK subunit is achieved by crossing mice with loxP-flanked IKKβ or IKK alleles with Nestin–Cre transgenic mice. The Cre recombinase under the control of the Nestin promoter mediates excision of loxP-flanked sequences in early neuronal precursors during embryonic life, resulting in target gene inactivation in nearly all neuroectodermal cells of the CNS, including neurons, astrocytes and oligodendrocytes. CNS-restricted elimination of NF-B activators IKKβ (IKKβCNS-KO mice) or IKK (IKKCNS-KO mice), but not IKK, ameliorates the pathology of murine autoimmune encephalitis (van Loo et al., 2006), suggesting that, at least in this disease model, canonical NF-B activation in cells of the CNS contributes to the severity of disease.
We inoculated IKKβCNS-KO, IKKCNS-KO and age-matched littermate controls carrying loxP-flanked (floxed) alleles but lacking expression of the Cre recombinase intracerebrally (i.c.) with 30 µl inoculum containing either a saturating (3x105 LD50) or a limiting (3x102 LD50) dose of the mouse-adapted prion Rocky Mountain Laboratory strain 6 (RML6; Falsig et al., 2008; Fig. 1a–c). Mice were maintained and monitored in accordance with the cantonal legislation for animal experiments in Switzerland. In parallel, we inoculated C57BL/6 animals as a further control.
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). The mean survival of IKKβCNS-KO mice after high-dose i.c. inoculation was 158±9 days (n=10), whereas the mean survival of the littermates without Nestin–Cre was 161±5 days (n=8; not significantly different to IKKβCNS-KO according to a two-tailed Mann–Whitney U-test; P=0.2949) and for wild-type (wt) control animals 153±4 days (n=4; P=0.0667). Similar results were found for mice inoculated with a low dose of prion inoculum with 194±9 days (n=11) for IKKβCNS-KO mice, 194±8 days (n=8; P=0.8518) for Nestin–Cre-negative littermates and 185±9 days (n=4; P=0.2828) for wt control animals. In line with these results, low-dose prion inoculation of IKKCNS-KO and control animals resulted in mean incubation periods of 187±8 days (n=8) for IKKCNS-KO mice and 193±5 (n=8; P=0.1139) days for Nestin–Cre-negative littermates (Fig. 1c). Again, the CNS-specific elimination of one further IKK subunit, IKK, did not exert any impact on prion pathogenesis in any of the mouse models used.
We then investigated brain sections of terminally sick mice for spongiform changes, activation of astrocytes, deposition of disease-specific PrP and activation of microglia (Fig. 1f–h). Histological analyses were performed as described previously (Sigurdson et al., 2006). No differences in spongiform changes, activation of astrocytes, deposition of disease-specific PrP or activation of microglia were detected among the IKKβCNS-KO and IKKCNS-KO mice and their respective littermates.
Next, brain homogenates were adjusted to 5 mg protein ml–1, and 50 µg total protein was separated by 12 % SDS-PAGE with optional pre-treatment with proteinase K (PK, 50 µg ml–1, 30 min, 37 °C). Proteins were transferred to nitrocellulose and membranes were blocked with Tris-buffered saline/0.1 % Tween 20/5 % non-fat milk, incubated with anti-PrP antibody POM1 (Polymenidou et al., 2005) and developed by enhanced chemiluminescence (Amersham). This series of experiments established that the abundance of PrPSc did not differ in brain homogenates of terminally sick IKKβCNS-KO, IKKCNS-KO and control animals (Fig. 1d, e).
We then investigated the efficiency of CNS-specific gene elimination by quantifying the abundance of IKKβ and IKK protein in brain homogenates (Fig. 2a–d). In accordance with published data (van Loo et al., 2006), we found approximately 60 % less IKKβ protein in the brains of IKKβCNS-KO animals and also approximately 60 % less IKK protein in IKKCNS-KO animals than in controls. These figures are likely to underestimate the extent of tissue-specific elimination in neuroectodermal cells, as the total brain homogenates contained a full range of CNS cells, including non-neuroectodermal elements such as microglia, which express high levels of the IKK signalling complex, yet do not express Nestin and would not be expected to recombine with the IKK loci.
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CNS-KO mouse model allowed the depletion of the canonical NF-B signalling pathway specifically in neuroectodermal cells, whereas other cell types including lymphocytes, macrophages, microglia and endothelial cells retained intact IKK signalling.
It was recently reported that the progression of experimental autoimmune encephalitis (EAE) was ameliorated in IKKβCNS-KO and IKKCNS-KO mice, which had only mild clinical signs of disease, less CNS inflammation and less tissue damage (van Loo et al., 2006). As CNS-specific reduction of IKKβ and IKK protein was sufficient to impair the course of EAE, one might conclude that NF-B activation in the CNS through the canonical and IKKβ- and IKK-dependent pathways is deleterious in autoimmune demyelinating diseases – possibly because non-microglial CNS cells (neurons, astrocytes and oligodendrocytes) amplify the inflammatory response in the CNS parenchyma by expressing pro-inflammatory mediators (van Loo et al., 2006).
In prion diseases, inflammatory infiltrates are modest and most certainly do not represent the primary cause of pathology, as completely immunodeficient mice develop scrapie, like wt mice (Klein et al., 1997). Accordingly, and in contrast to the EAE studies discussed above, the results reported here exclude a role for neuroectodermal IKKβ or IKK, and therefore for the canonical NF-B signalling pathway, in prion pathogenesis. However, it cannot be excluded that microglia-borne IKKβ or IKK may be involved in prion pathogenesis, as the microglial compartment does not experience gene elimination in Nestin–Cre mice.
In contrast to global elimination of the β or subunit of the IKK signalosome, prevention of IKK phosphorylation does not induce early lethality in mice, as reported for a mouse strain expressing a non-phosphorylatable IKKAA knock-in allele whose activation loop contained alanines in place of the phosphoacceptor serine (Cao et al., 2001). Apart from a severe lactation defect due to impaired proliferation of mammary epithelial cells in females, IKKAA/AA mice are healthy and fertile. To assess the impact of the alternative (non-canonical) NF-B pathway in prion disease, we inoculated IKKAA/AA and IKKwt/AA mice i.c with high (3x105 LD50) or low (3x102 LD50) doses of RML6 prions. As the IKKAA/AA mice were derived from a 129/C57BL/6 mixed background, we crossed them with 129/C57BL/6 F1 progeny to produce IKKwt/AA mice. In contrast to inoculation experiments performed with IKKβCNS-KO and IKKCNS-KO mice, we observed a marginally significant (P=0.027) reduction in the disease period of 26 days after i.c. inoculation of a high dose of prions. However, the IKKAA/AA survival curve appeared to be broadly distributed and showed a mean survival period of 127±19 days (n=10) compared with 153±12 days (n=5) for IKKwt/AA mice (Fig. 3a, b). This difference was reduced to 15 days after low-dose i.c. inoculation. Although a trend was observed, the survival curves did not differ significantly (P=0.1453). Whereas mean survival of IKKAA/AA mice was 167±21 days (n=9), in IKKwt/AA mice this was 182±11 days (n=15). We concede that the mixed genetic background of the animals used here may represent a confounding factor.
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AA/AA and IKKwt/AA mice (Fig. 3a, b, right panels). In addition, we could not observe differences in the abundance of PrPSc in brain homogenates of terminally sick IKKAA/AA and IKKwt/AA mice as determined by Western blot analysis (Fig. 3c).
Consequently, our studies utilizing the IKKAA/AA mouse model do not support a role for the alternative NF-B signalling pathway in prion disease. Importantly, several observations lend support to our interpretation of a lack of involvement of IKK in prion pathogenesis: (i) the very small difference in incubation period; (ii) the broad distribution of the IKKAA/AA survival curve compared with the control group; and (iii) the fact that the groups inoculated with a low dose of prions did not differ in disease period. Similar to our findings in prion disease, deletion of IKK did not impair the progress of EAE (van Loo et al., 2006).
In summary, we have been unable to gather any evidence suggesting that NF-B signalling influences the manifestation of prion disease in a variety of mouse models. The finding that depletion of either classical or alternative NF-B signalling in neuroectodermal cells of the CNS, including neurons, astrocytes and oligodendrocytes, does not impair prion pathogenesis in the brain is surprising, as the NF-B signalling pathways represent crucial triggers in proliferation, induction of inflammation, regulation of apoptosis and immune responses involving induction of inflammation. It has been claimed that NF-B activity leads to mitochondrial apoptosis after prion infection (Bourteele et al., 2007). However, it should be noted that in the latter study, Nfkb2–/– and Bcl-3–/– mice showed only a mild reduction of 11 and 15 days in disease progression after prion inoculation, whereas Nfkb1–/– and p65CNS-KO animals behaved in a similar way to control animals. When viewed in the context of the results reported here, even this former study could be interpreted as suggesting that NF-B signalling is not a major determinant of prion pathogenesis.
Despite much effort in prion research, the mechanisms of neuronal loss are still elusive (Aguzzi et al., 2007). Understanding the molecular and cellular underpinnings of neuronal loss during prion disease could help in the development of possible treatments. Furthermore, knowledge of the impact of protein aggregates on neuronal cell survival could shed light on other protein-aggregation diseases such as Alzheimer's and Parkinson's disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 November 2007;
accepted 15 February 2008.
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