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The Journal of Neuroscience, September 15, 2004, 24(37):7999-8008; doi:10.1523/JNEUROSCI.2675-04.2004
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Neurobiology of Disease
Activation of the I B Kinase Complex and Nuclear Factor-B Contributes to Mutant Huntingtin Neurotoxicity
Ali Khoshnan,1
Jan Ko,1
Erin E. Watkin,1
Lisa A. Paige,2
Peter H. Reinhart,2 and
Paul H. Patterson1
1Biology Division, California Institute of Technology, Pasadena, California 91125, and 2Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710
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Abstract
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Transcriptional dysregulation by mutant huntingtin (Htt) protein has been implicated in the pathogenesis of Huntington's disease (HD). We find that cultured cells expressing mutant Htt and striatal cells from HD transgenic mice have elevated nuclear factor- B (NF-B) activity. Furthermore, NF-B is concentrated in the nucleus of neurons in the brains of HD transgenic mice. In inducible PC12 cells and in HD transgenic mice, mutant Htt activates the IB kinase complex (IKK), a key regulator of NF-B. Activation of IKK is likely mediated by direct interaction with mutant Htt, because the expanded polyglutamine stretch and adjacent proline-rich motifs in mutant Htt interact with IKK , a regulatory subunit of IKK. Activation of IKK may also influence the toxicity of mutant Htt, because expression of IKK promotes aggregation and nuclear localization of mutant Htt exon-1. Moreover, in acute striatal slice cultures, inhibition of IKK activity with an N-terminally truncated form of IKK blocks mutant Htt-induced toxicity in medium-sized spiny neurons (MSNs). In addition, blocking degradation of NF-B inhibitors with a dominant-negative ubiquitin ligase  -transducin repeat-containing protein also reduces the toxicity of mutant Htt in MSNs. Therefore, aberrant NF-B activation may contribute to the neurodegeneration induced by mutant Htt.
Key words: Huntington's disease; IKK; polyproline; striatum; polyQ; intrabody
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Introduction
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Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of polyglutamine (polyQ) in exon-1 (HDx1) of huntingtin (Htt) (Huntington's Disease Collaborative Research Group, 1993 ). Neuronal death in HD has been variously attributed to polyQ toxicity, activation of caspases, interference with transcription, and sequestration of cellular proteins (Li, 1999; Tobin and Signer, 2000; Wanker, 2000; Zoghbi and Orr, 2000). Several proteins that interact with HDx1 have been identified (Cattaneo et al., 2001), and although the function of some of these proteins in the etiology of HD is unclear, the transcriptional coactivator cAMP response element (CRE)-binding protein (CBP), as well as proteins containing WW domains, have been implicated in HD pathology (Passani et al., 2000; Steffan et al., 2000; Nucifora et al., 2001). Htt binding blocks CBP-mediated gene expression, and histone deacetylase inhibitors reduce polyglutamine-induced cell death in Drosophila (Steffan et al., 2001). Conversely, CRE-mediated gene expression, which requires CBP, is enhanced in HD transgenic mice (Obrietan and Hoyt, 2004). CBP participates in many signaling pathways, including NF-B (Chen et al., 2002; Zhong et al., 2002). Consequently, mutant Htt dysregulation of CBP can influence nuclear factor-B (NF-B)-mediated gene expression.
NF-B is sequestered in the cytoplasm by a family of inhibitory proteins (IBs) (Ghosh et al., 1998). IBs are phosphorylated by a signal-activated kinase complex known as I-B kinase (IKK) (Ghosh and Karin, 2002). This complex contains two catalytic subunits, IKK and IKK, and a regulatory module, IKK (Karin and Lin, 2002). Phosphorylated IBs are ubiquitinated by an F-box E3-ligase, -transducin repeat-containing protein (-TrCP) (Spencer et al., 1999) and subsequently degraded by proteosomes. Liberated NF-B can bind DNA and promote gene expression (Pahl, 1999).
In the nervous system, cytokines, neurotrophic factors, neurotransmitters, injury, seizure, and proteins implicated in neurodegenerative disorders can all activate the NF-B pathway (Mattson and Camandola, 2001). Injection of 3-nitropropionic acid (3NP) and kainate into the striatum, which mimics pathology caused by mutant Htt, results in activation of NF-B and expression of the proapoptotic gene products, myc and p53 (Qin et al., 1999). On the other hand, injection of 3NP into the striatum of mice lacking the p50 subunit of NF-B results in diminished NF-B activity and increased cell death (Mattson and Camandola, 2001).
Akt/protein kinase B, an activator of IKK, has been shown to phosphorylate mutant Htt and appears to have a neuroprotective effect (Datta et al., 1999; Humbert et al., 2002). Furthermore, striatal cells from HD knock-in (KI) mice display elevated Akt activity; however, expression of mutant Htt in these cells is not toxic (Gines et al., 2003). Akt also phosphorylates ataxin-1, which binds to 14-3-3 protein and enhances its stability, aggregation, and neurotoxicity (Chen et al., 2003; Emamian et al., 2003). Thus, as a major integrator of cell signaling, NF-B may regulate the pathogenesis of polyglutamine-induced neurodegeneration. Here we show that mutant Htt binds IKK, activates the IKK complex, and elevates NF-B-dependent gene expression. Furthermore, HDx1-mediated toxicity is significantly reduced by genetic inhibition of IKK and NF-B activity.
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Materials and Methods
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Cells and transgenic animals. PC12 cells, which express wild-type (WT) HDx1-enhanced green fluorescent protein (EGFP) with 25 glutamine repeats (25Qs) or mutant HDx1-EGFP with 103Qs in response to ecdysone, were kindly provided by Dr. E. Schweitzer (University of California Los Angeles). These cells were cultured on a collagen I substrate (Fisher Scientific, Tustin, CA) in DMEM with 5% horse serum, 5% fetal bovine serum, 2 mM glutamine, and standard penicillin-streptomycin antibiotics. Immortalized striatal cells from WT and HD KI mice, provided by Dr. M. E. MacDonald (Massachusetts General Hospital, Boston, MA), were cultured as described (Gines et al., 2003). Human embryonic kidney (HEK)-293 cells were cultured as described previously (Khoshnan et al., 2002). The HD transgenic mouse line R6/2 and a knock-in line, in which a 155 polyglutamine stretch was inserted in HDx1 of mouse Htt, have been described (Davies et al., 1997; Lin et al., 2001). Colonies were established and maintained in our animal facility.
Gene reporter assays and transfections. The pNF-B-luciferase with five enhancer elements and the control plasmid without NF-B binding sites, pCIS-CK-luciferase, were used for gene reporter assays (Stratagene, La Jolla, CA). To normalize for transfection efficiency between samples, a -galactosidase (-gal) construct expressed from the elongation factor (EF)-1 promoter (Invitrogen, Carlsbad, CA) was included in all gene reporter assays. PC12 cells were transfected with control or NF-B-luciferase plasmids using lipofectamine-plus. On the following day, cells were left untreated or stimulated for 8 hr with 1 µg/ml ecdysone to induce expression of HDx1. Mock treatment or NGF (Sigma, St. Louis, MO) was added (50 ng/ml) and incubated for an additional 6 hr. Cells were suspended in lysis buffer on ice for 10 min and cleared by centrifugation. Luciferase activity was measured by addition of the substrate (Promega, Madison, WI) to equal amounts of protein from each sample. Striatal cells from WT and HD KI mice were transfected with an NF-B reporter plus -gal and anti-polyQ (MW2) or anti-polyproline [antipolyP (MW7)] recombinant intrabodies (Khoshnan et al., 2002). On the following day, cells were starved for 2 hr and treated with 5 ng/ml recombinant interleukin (IL)-1 (R & D Systems, Minneapolis, MN) and incubated for an additional 6 hr. Cells were harvested and cleared lysate was used to measure luciferase as described above. All gene reporter assays were corrected for transfection efficiency by normalizing the units of -gal in the extracts, using the -gal assay (Invitrogen). -gal values from the samples were divided by the -gal value from control, 25polyQ, noninduced PC12 cells (no expression of HDx1) and multiplied by each corresponding luciferase reading. For striatal cells, -gal values were divided by the value obtained for WT cells without any treatment. Results are shown as relative luciferase units and are representative of at least three independent experiments. Data points are the average of triplicate measurements.
To examine the effects of deleted F-box ( F)-TrCP [obtained from Dr. R. Deshaies (California Institute of Technology) and originally provided by Spencer et al. (1999)] and deleted N terminus (DN)-IKK on mutant HDx1-induced NF-B, PC12 cells were transfected with F-TrCP or DN-IKK plus -gal and NF-B reporter in six-well plates. Induction of HDx1 with ecdysone and NGF treatment was as described above. Equal amounts of each sample were used to measure luciferase activity. Binding of NF-B to its consensus oligonucleotide site was assayed in striatal neurons with the BD TransFactor Kit specific for the p65 subunit following the manufacturer's instructions (BD Biosciences, Mountain View, CA). Briefly, striatal cell lines from WT and HD KI mice were starved for 2 hr and treated with 5 ng/ml IL-1 for 15 or 30 min. Nuclear extracts were obtained using commercial reagents (BD Biosciences). Protein concentration was determined by the BCA method (Pierce, Rockford, IL). Equal amounts of each nuclear fraction were added to wells coated with an NF-B consensus or mutated control oligonucleotide and incubated at room temperature for 1 hr. Wells were washed extensively and incubated with primary rabbit antibody specific for the p65 subunit of NF-B. Bound p65 was detected with a goat anti-rabbit antibody conjugated to horseradish peroxidase. After addition of the substrate, 3,3-5,5-tetramethylybenzidine, color development was measured at 655 nm using an ELISA plate reader. Samples were done in triplicate, and the results shown are representative of three independent experiments.
Transfection of HEK-293 cells with mutant HDx1-EGFP alone (Kazantsev et al., 1999) or with one of the following HA-tagged constructs, full-length (F), DN, and deleted C terminus (DC) of IKK (provided by Dr. E. Zandi, University of Southern California) (Rothwarf et al., 1998) or F-TrCP, was done using lipofectamine-plus reagents (Invitrogen). For histochemistry, cultured cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 70% methanol for 1 hr, and washed with PBS. Cells were stained with anti-HA tag for IKK or anti-myc for F-TrCP (Cell Signaling, Beverly, MA) followed by a goat anti-mouse antibody conjugated to Alexa 594 (Molecular Probes, Eugene, OR). Toto-3, a dimeric cyanine, was used to stain nuclei (Molecular Probes). Cells were washed in PBS, mounted on microscope slides, and examined with a confocal microscope. Mutant HDx1 toxicity was assessed by counting the number of condensed GFP-positive bodies, which are remnants of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive (TUNEL+) cells (Khoshnan et al., 2002), using a florescence microscope. For TUNEL staining, transfected cells grown on coverslips were air dried and fixed 16 hr after transfection as above and permeabilized in ethanol/acetic acid (2:1). Apoptotic cells were labeled with terminal deoxynucleotidyl transferase using digoxigenin-labeled nucleotides and detected by anti-digoxigenin antibody and rhodamine-conjugated secondary according to the instructions provided by the manufacturer (Chemicon, Temecula, CA).
Immunohistochemistry of tissue sections. Brains were taken from paraformaldehyde-fixed WT and R6/2 HD transgenic mice, and cryosections were permeabilized in 70% methanol for 1 hr at -20. After blocking in 3% BSA and 10% normal goat serum, slides were incubated with anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA) and the neuronal marker anti-neuronal-specific nuclear protein (NeuN) antibodies (Chemicon). p65-positive cells were detected with a goat anti-rabbit secondary antibody conjugated to FITC and goat anti-mouse Alexa Fluor 594 (Molecular Probes). Toto-3 was used to stain nuclei. Sections were examined with a confocal microscope. Total neurons from 16 coronal sections containing cortical and striatal areas of four animals each of WT and HD mice were quantified, and the average percentage of cells with nuclear p65 is presented.
Protein-protein interaction. In vitro binding of IKK to mutant HDx1 was performed using glutathione S-transferase (GST)-pull down assays. GST-HDx1 with 20 or 51 polyQs, with and without polyproline domains, was obtained from Dr. E. Wanker (Max Plank Institute, Berlin, Germany). These constructs were expressed in Escherichia coli and purified with glutathione beads (Amersham Biosciences, Piscataway, NJ) according to manufacturer's instructions. IKK constructs were transcribed and translated in rabbit reticulocytes in the presence of [35S]methionine (Promega). Briefly, 5 µg of GST-HDx1 bound to beads was incubated with 10 µl of each labeled in vitro-translated IKK product in 500 µl of Tris-based buffer containing 10% glycerol and 5 mM DTT and rocked at room temperature for 2 hr. Beads were washed five times in the same buffer. Bound IKK was examined by SDS-PAGE, followed by autoradiography. Recombinant anti-HDx1 intrabodies (Khoshnan et al., 2002) were expressed and examined similarly for binding to GST-HDx1. For competition assays, 10-fold excess in vitro-translated intrabodies were added to GST-HDx1 plus DC-IKK and processed as above.
For coimmunoprecipitation studies, PC12 cells or striatal tissue of WT and HD transgenic mice was lysed by sonication in buffer A (50 mM HEPES, pH 7.6, 250 mM NaCl, 1% Triton X-100, 2 mM MgCl2, 2 mM DTT, 1 mM Na3VO4, and 20 µM -glycerophosphate) and a mixture of protease inhibitors (Boehringer Mannheim, Mannheim, Germany). Equal amounts of cleared extracts from each sample were incubated with a rabbit anti-IKK (Santa Cruz Biotechnology) coupled to protein-A beads and rocked for 3 hr at 4°C. Cells were washed five times with buffer A. Immune complexes were examined by SDS-PAGE followed by Western blotting with monoclonal antibodies targeting Htt (Ko et al., 2001), as described in the figure legends.
Immune complex kinase assays. To measure IKK activity, immunoprecipitated IKK complexes were examined for the ability to phosphorylate GST-IB in vitro. The construct for the N-terminal 61 amino acids of GST-IB, which contains the IKK phosphorylation sites (provided by Dr. W. Greene, University of California San Francisco), was expressed in E. coli and purified on glutathione beads as described in the manufacturer's instructions (Amersham Biosciences). To obtain IKK complexes, equal amounts of cleared striatal extracts from WT and HD animals, or from PC12 cells induced to express WT or mutant HDx1-EGFP (all in buffer A), were incubated with a mouse anti-IKK antibody (Santa Cruz Biotechnology) coupled to protein-A beads and rocked for 3 hr at 4°C. Beads were washed five times in buffer B (50 mM HEPES, pH 7.6, 250 mM NaCl, 1 M urea, 0.1% Nonidet P-40, 6 mM EDTA, 6 mMEGTA, 1 mM DTT with 20 µM -glycerophosphate) and a mixture of protease inhibitors and equilibrated in kinase buffer (20 mM HEPES, pH 7.6, 2 mM MgCl2, 2 mM MnCl2, 10 µM ATP, 10 mM glycerophosphate, 10 mM NaF, 300 µM Na3VO4, 1 mM dithiothreitol DTT). IKK activity was evaluated in vitro with 1 µg of purified GST-IB (N-terminal 61 amino acids) in the presence of 10 µCi of [32P]-ATP for 30 min at 30°C. Products were examined by SDS-PAGE followed by autoradiography. Duplicate samples were examined by Western blotting using a rabbit anti-IKK antibody (Santa Cruz Biotechnology).
Brain slice preparation. All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee and Duke University Medical Center Animal Guidelines. Postnatal day 7 (P7) CD Sprague Dawley rats (Charles River Laboratory, Raleigh, NC) were decapitated, and the brains were surgically removed and placed in ice-cold Neurobasal medium (Invitrogen). The tissue was fixed to a chilled, stainless steel Vibratome stage using cyanoacrylate glue (Krazy-Glue) and covered with Neurobasal medium. Coronal brain slices (250 µm thick) were cut by Vibratome (VT1000S; Leica, Nussloch, Germany) as described previously (Edgerton and Reinhart, 2003). Brain slices were kept at 37°C in 5.0% CO2 for 1 hr.
Biolistic transfection. Gold particles (1.6 µm gold microcarriers; Bio-Rad, Hercules, CA) were used as DNA carriers for transfection as described previously (Lo et al., 1994). Briefly, gold particles were sonicated in 0.05 M spermidine in the presence of plasmid DNA. The gold-DNA mixture was washed three times in 100% ethanol before being loaded into Helios plastic cartridges (Bio-Rad) according to manufacturer's instructions. Slices were transfected using a Bio-Rad Helios gun with a cyan fluorescent protein (CFP)-tagged Htt fragment, together with yellow fluorescent protein (YFP) as a morphometric marker. In a number of experiments, one of the IKK constructs was also cotransfected with the Htt-construct and YFP. CFP-tagged Htt fragments were exon-1 N-terminal fragments containing either a short-Q (Q23) or a long-Q (Q148) polyQ domain.
Brain slice neurodegeneration assay. For each condition, transfections were performed on 12 brain slices per experiment. Protein expression and neurodegeneration (loss of processes, shriveling of the soma) were assayed 2-7 d after transfection using a Leica fluorescence microscope with appropriate filters for YFP and CFP. The total area of the striatum was identified, and transfected MSNs were identified by their morphology. CFP fluorescence was used to determine expression of Htt fragments and their aggregation into macro-inclusions. Each experiment involved 3-12 transfected brain slices per condition, and the data are means from six independent experiments for each condition.
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Results
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Mutant Htt activates the NF-B pathway
We first used PC12 cells that express WT (25Q) or mutant (103Q) HDx1 in response to ecdysone (Fig. 1A). These cells were transfected with a reporter construct containing NF-B enhancer element fused to luciferase. The inducible nature of HDx1 expression allows the examination of soluble mutant HDx1 function, before macro-aggregates become visible. Induction of mutant HDx1 expression increases transcription from the NF-B-dependent promoter approximately fivefold over that of noninduced cells, whereas WT HDx1 has minimal effect (Fig. 1A). These results are consistent with a recent report demonstrating upregulation of NF-B expression by mutant HDx1 in inducible PC12 cells (Sugars et al., 2004). To examine whether mutant HDx1 also influences signal-induced NF-B activation, we used NGF, which is known to activate NF-B in PC12 cells (Foehr et al., 2000). Mutant (but not WT) HDx1 strongly enhances NGF-induced NF-B-dependent gene expression (Fig. 1A). Because mutant Htt has been shown to variably influence gene expression from different promoters, we performed the same experiments with a control plasmid that lacks an NF-B enhancer element. Expression of WT or mutant HDx1 does not influence luciferase expression from the control plasmid (Fig. 1B), suggesting that the elevated NF-B values in Figure 1A are mediated by mutant HDx1 expression. Under the conditions tested, most of the HDx1 remains in the detergent-soluble fraction and is detectable by Western blotting (Fig. 1A); however, the NF-B activity diminishes as mutant HDx1 accumulates and aggregates (data not shown). Thus, soluble mutant HDx1 activates endogenous, and augments NGF-induced, NF-B-dependent gene expression. Moreover, expression of mutant HDx1 promotes degradation of the inhibitory protein IB, a hallmark of NF-B activation. Levels of IB drop significantly by 3 hr and return to normal by 7 hr after induction (Fig. 1C). These data confirm the gene reporter assay results and suggest that mutant Htt-induced NF-B is mediated by degradation of IB.
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Figure 1. Mutant Htt activates NF-B-dependent gene expression. A, PC12 cells transfected with an NF-B reporter plasmid were induced to express a 25 or 103 polyQ-containing HDx1-EGFP. NGF was added 8 hr after Httinduction, and the cells were incubated for an additional 6 hr. Cleared lysates were assayed for luciferase activity to assess NF-B-dependent gene expression. The luciferase values were normalized for transfection efficiency using the -gal reporter gene under transcriptional control of the EF-1 promoter. -gal values from samples were divided by the -gal values from control, 25polyQ, noninduced HDx1 (no expression of HDx1) and multiplied by the corresponding luciferase readings. Results are shown as relative luciferase units and are representative of at least three independent experiments, and data points are the average of triplicate measurements. The Western blot at the bottom of A shows the level of soluble HDx1 detected using an anti-Htt antibody. The same blots were probed with anti-tubulin to show equivalent loading in each lane. B, Expression of WT or mutant HDx1 does not influence luciferase expression from the control plasmid, which lacks an NF-B enhancer element. The experiment was done as in A, using a plasmid reporter without the NF- Benhancers. Luciferase units were  100-fold less than the activity observed with the NF-B reporter. C, NF-B activation by mutant Htt includes degradation of IB. PC12 cells were treated with ecdysone to induce HDx1 for the indicated time and examined by Western blotting with anti-IB antibody. The same membrane was stained with anti-tubulin antibody for loading accuracy. D, Full-length mutant Htt enhances IL-1-mediated NF-B activation. Striatal cells from WT and HD KI mice were transfected with NF-B or the control reporter and the indicated plasmids. Transfected cells were starved on the following day for 2 hr and treated with 5 ng/ml IL-1 for an additional 6 hr. Luciferase activity was measured in equal amounts of cleared lysate from each sample and normalized to -gal units from WT striatal cells without IL-1.
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To examine whether full-length mutant Htt also influences NF-B activity, we used immortalized striatal cell lines obtained from WT and HD KI mice (Gines et al., 2003). Using the reporter with NF-B enhancer, no significant difference in basal NF-B activity is observed between striatal cells from WT and HD KI mice (Fig. 1D) (compare C lanes in WT and mutant). These data are consistent with a recent report showing that inducible expression of full-length WT or mutant Htt in PC12 cells has no effect on basal NF-B activation (Sugars et al., 2004). We therefore asked whether WT and mutant cells respond differently to NF-B-inducing agents. KI striatal cells display a stronger response than WT cells to the NF-B-inducing cytokine IL-1 (Fig. 1D). To verify that mutant Htt mediates the enhanced response to IL-1, we used recombinant single chain intrabodies, which are known to interfere with the function of mutant HDx1 (Khoshnan et al., 2002) (see below). Two intrabodies, MW2 targeting the expanded polyQ and MW7 recognizing the polyP motifs of HDx1, reduce the IL-1-mediated NF-B activation in striatal cells from KI mice (Fig. 1D). MW7 is somewhat more potent than MW2 at inhibiting the effects of full-length mutant Htt on NF-B.
Activated NF-B functions primarily in the nucleus, where it regulates gene expression. To confirm the authenticity of the gene reporter assays, we measured nuclear p65 binding to a consensus NF-B oligonucleotide. Extracts from mutant Htt KI striatal cells show elevated nuclear p65 binding in response to IL-1 treatment when compared with the equivalent samples from WT striatal cells (Fig. 2A). Specificity of binding was confirmed using a mutated consensus NF-B oligonucleotide, which failed to show p65 binding in this assay (data not shown). Thus, cells expressing full-length mutant Htt respond more vigorously than cells with WT Htt to IL-1-induced NF-B nuclear localization. We also examined whether there is elevated nuclear NF-B in HD mouse brains. Brain sections from 8-week-old R6/2 HD mice and age-matched controls were stained with an antibody that recognizes the p65 subunit. p65 is concentrated in the nucleus of a majority of the NeuN-positive neurons in the cortex and striatum of HD mice, whereas most of the neurons of the age-matched WT mice contain cytoplasmic p65 (Fig. 2B,C). Thus, mutant Htt promotes nuclear localization of NF-B in vivo as well as in cell culture.
Mutant Htt activates the IKK complex
Signal-induced phosphorylation of serine residues 32 and 36 of IB, which is mediated by the IKK complex, is essential for the ubiquitination and proteosome-mediated degradation of IB (Ghosh and Karin, 2002). Because IB degradation is promoted with mutant HDx1 induction, we examined whether the IKK complex is activated by mutant HDx1 expression. Immunoprecipitated endogenous IKK complexes from extracts of control or PC12 cells induced to express mutant HDx1 were analyzed for their kinase activity by measuring phosphorylation of the recombinant substrate, GST-IB. The kinase activity of IKK from cells expressing mutant HDx1 is significantly higher than that from non-HDx1-induced cells (Fig. 3A). As expected, NGF treatment also activates the IKK complex, and consistent with our gene reporter assay results, kinase activity appears to be elevated somewhat further when both NGF and HDx1 are present (4.1-fold compared with 2.4- and 3.2-fold in the ecdysone and NGF-treated cells, respectively). Expression of WT HDx1 has no effect on IKK activity (data not shown).
To investigate whether IKK activation by mutant HDx1 also occurs in vivo, we used two lines of HD transgenic mice, R6/2 (Davies et al., 1997) and a mutant Htt KI (Lin et al., 2001). IKK complexes immunoprecipitated from striatal or cortical extracts of WT and HD mice were assayed in vitro for IKK activity. Consistent with the PC12 cell results, IKK activity is higher in complexes isolated from striatal (9-fold) as well as cortical (3.8-fold) extracts of 2-month-old R6/2 mice compared with age-matched WT controls (Fig. 3B). With KI HD mice, a significant increase in IKK activity is detected in the striatum at 1 year of age (4.8-fold higher than WT) (Fig. 3C).
Because IKK activity is elevated in cell culture and in animal models expressing mutant Htt, we tested whether HDx1 associates with IKK. Complexes isolated with an anti-IKK antibody from PC12 cells were examined for the presence of HDx1 by Western blotting. Soluble mutant HDx1 coprecipitates with IKK, whereas minimal WT HDx1 is observed (Fig. 3D). Thus, IKK physically forms a complex with mutant HDx1. This interaction also occurs in the brain of HD mice. IKK complexes isolated from brain extracts of R6/2 mice contain soluble HDx1, which is recognized by anti-Htt antibody (Fig. 3E). Therefore, activation of IKK by mutant HDx1 may be, in fact, the result of Htt binding to the complex.
Mutant Htt interacts with IKK
The activity of the IKK complex is regulated by IKK, a glutamine-rich nonkinase subunit of the complex (Rothwarf et al., 1998; Ghosh and Karin, 2002). A homolog of IKK that participates in tumor necrosis factor- receptor signaling interacts with Htt in a yeast two-hybrid assay (Hattula and Paranen, 2000). Therefore, we asked whether the binding of mutant HDx1 to the IKK complex is mediated by IKK. A GST pull-down assay using HDx1 fused to GST and [35S]methionine-labeled IKK was used. The stringency of the assay was such that there was no binding to GST alone. In vitro translated-IKK preferentially binds to GST-HDx1 containing expanded polyQ (Fig. 4A). Although deletion of the C-terminal 119 amino acids of IKK (DC-IKK) has no apparent effect on this binding, removal of the first 134 amino acids (DN-IKK) diminishes binding to HDx1 (Fig. 4A). Interestingly, mutant HDx1 without its polyP domains does not bind efficiently to IKK (Fig. 4B). Although we cannot rule out that the absence of proline may alter the solubility of mutant HDx1 and thus artifactually reduce binding to IKK, the importance of the polyP domains is further supported by antibody experiments. We performed competition assays using in vitro-translated, anti-Htt recombinant intrabodies (Khoshnan et al., 2002). Coincubation of DC-IKK plus mutant HDx1 with 10-fold excess of intrabodies specific for polyQ (MW2) or polyP (MW7) competes with binding of IKK to HDx1 (Fig. 4B). Moreover, these intrabodies inhibit the HDx1-induced NF-B-dependent gene expression in PC12 cells (Fig. 4C). Although anti-polyQ intrabody has a modest inhibitory effect, anti-polyP (MW7) significantly minimizes mutant HDx1-induced NF-B activation. Thus, IKK binding to mutant HDx1 requires the polyQ expansion and is influenced by the polyP domains. Moreover, this interaction likely plays a role in mutant HDx-1-induced IKK and NF-B activation.
Inhibition of the NF-B pathway reduces HDx1-induced toxicity in cell culture and brain slices
Because binding of mutant HDx1 to IKK influences IKK activity and NF-B-mediated transcription, it was important to determine whether IKK can modify the toxicity of mutant HDx1. Confocal microscope examination of HEK-293 cells expressing mutant HDx1 and IKK at an early time point before widespread toxicity shows that full-length IKK promotes aggregation of mutant HDx1 (Fig. 5A). Intracellular, microscopically visible aggregates are apparent, and importantly, some of these aggregates localize in the nucleus of transfected cells. C-terminally truncated IKK, which also binds to mutant HDx1 (Fig. 4 A), has a similar effect (data not shown). On the other hand, DN-IKK, which does not bind to mutant HDx1, has no apparent effect on HDx1 aggregation (Fig. 5A). Thus, binding to IKK appears to promote aggregation and nuclear localization of mutant HDx1. Overexpression of full-length IKK appears to have little effect on toxicity at 48 hr after transfection, however, because the overall number of cells killed by mutant HDx1 does not appear to change (Fig. 5A). Toxicity was assessed by counting the number of condensed GFP+ bodies at 48 hr after transfection, which are remnants of dead cells (Khoshnan et al., 2002) (see Fig. 7B). Importantly, DN-IKK strongly reduces the toxicity of mutant HDx1 (Fig. 5A, graph). The effect of DN-IKK on mutant HDx1 toxicity was confirmed by TUNEL assay. Compared with cultures with or without full IKK, the number of TUNEL+ cells is significantly reduced in cells cotransfected with DN-IKK and mutant HDx1 (Fig. 5B). DN-IKK lacks the binding domain essential for interaction with IKK and - and interferes with IKK activity (May et al., 2000; Poyet et al., 2000). DN-IKK also blocks basal and NGF-induced NF-B-dependent gene expression by mutant HDx1 in inducible PC12 cells (Fig. 5C). Therefore, inhibition of mutant HDx1 toxicity by DN-IKK may be mediated through blockage of the NF-B pathway.
To extend these results to a more in vivo-like model, we used a novel, acute, P7 rat brain slice assay. A biolistic method is used to cotransfect neurons with three constructs: HDx1-CFP to visualize HDx1 expression and associated neurodegeneration, YFP to monitor the full morphology of transfected cells, and IKK or DN-IKK. Individual, transfected MSNs are identified by location and morphology and monitored daily for 7 d after transfection. Neuronal viability was quantified by counting the number of CFP+ neurons in each slice at 7 d. Expression of mutant HDx1 leads to the formation of Htt aggregates (shown in cyan) 1-2 d after transfection and to cell death within 4-7 d. YFP apoptotic bodies are seen initially, followed by complete loss of YFP fluorescence. In contrast, neurons transfected with WT HDx1 do not contain Htt inclusions and retain their normal dendritic structure (Fig. 6A). Coexpression of full-length IKK or DN-IKK has no obvious effect on MSNs expressing WT HDx1 (Fig. 6A,B). Furthermore, expression of IKK has no significant effect on neurodegeneration induced by expression of mutant HDx1 (Fig. 6A,B); however, the coexpression of DN-IKK with mutant HDx1 is significantly neuroprotective (Fig. 6A,B). The neuroprotective function of DN-IKK is essentially independent of aggregate formation by mutant Htt, because rescued neurons still contain Htt macro-aggregates (Fig. 6A). These brain slice results indicate that DN-IKK also reduces the neurotoxicity of mutant HDx1 in MSNs residing in a more intact, three-dimensional setting.
Because ubiquitination of IB is a crucial step in liberating NF-B, we predicted that blocking this step should have an effect similar to that observed with DN-IKK. The E3-ubiquitin ligase TrCP specifically promotes degradation of IB (Spencer et al., 1999). A dominant-negative form of TrCP (F-TrCP), which has been shown to block degradation of phosphorylated IBs, was cotransfected with the NF-B reporter in PC12 cells. Expression of F-TrCP abolishes basal and mutant HDx1-induced NF-B activity (Fig. 7A). As predicted, F-TrCP expression also blocks the toxicity of mutant HDx1 in cultured cells (Fig. 7B,C). Cells expressing F-TrCP and mutant HDx1 remain intact, whereas cells transfected with mutant HDx1 and control vector become condensed and eventually detach from the culture dish (Fig. 7B, arrow). Moreover, expression of F-TrCP in the brain slice assays significantly reduces the toxicity of mutant HDx1 in MSNs (Fig. 7D). WT-TrCP does not influence the toxicity of mutant HDx1 in MSNs (Fig. 7D). Collectively, these data show that blocking IKK activity or degradation of IB, both of which result in inhibition of NF-B, can reduce the toxicity of mutant HDx1.
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Discussion
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Transcriptional dysregulation has been implicated in HD pathology, and mutant Htt can sequester transcription factors and reduce gene expression (Cattaneo et al., 2001; Ross, 2002; Yu et al., 2002) or increase the transcriptional activity of specific signaling pathways (Yohrling et al., 2003; Obrietan et al., 2004). We find that soluble mutant Htt interacts with the IKK complex, promotes phosphorylation and degradation of IB, and subsequently enhances NF-B activity. Results from the HD cell culture model are supported by findings that IKK complexes from striatal and cortical extracts of HD transgenic mice also have elevated kinase activity, and activated NF-B is concentrated in the nuclei of cortical and striatal neurons of HD transgenic mice. Most importantly, interference with the NF-B pathway by expression of DN-IKK or F-TrCP reduces the toxicity of mutant Htt in cell culture and protects striatal MSNs in a brain slice model of HD. These findings implicate the NF-B pathway in the neurotoxicity of mutant Htt.
Current paradigms of HD pathology include increased sensitivity to NMDA-mediated excitotoxicity and activation of caspases (Cepeda et al., 2001; Zeron et al., 2002). Although the signaling cascades that contribute to enhanced sensitivity of striatal neurons in HD are poorly understood, derangement of Ca2+ efflux from the endoplasmic reticulum by binding of mutant Htt to the inositol (1,4,5)-triphosphate receptor may be a contributing factor (Tang et al., 2003). In neurons and other cell types, elevated Ca2+ influx is a major activator of the IKK complex and subsequently NF-B (Lilienbaum and Israel, 2003). We find that striatal cells from HD KI mice expressing full-length mutant Htt respond more vigorously to IL-1-induced NF-B activation (Fig. 1C,D). IL-1 can increase NMDA-mediated neurotoxicity by activation of tyrosine kinases and subsequent phosphorylation of NMDA receptor (Viviani et al., 2003). It is relevant that brain lysates of R6/2 HD mice have significantly higher levels of IL-1 than WT controls (Ona et al., 1999). In addition, inhibitors of IL-1 converting enzymes are effective against the toxicity of mutant Htt in tissue culture and animal models (Chen et al., 2000). Mutant HDx1 also enhances NGF-induced IKK and NF-B activation (Figs. 1A, 3A). Therefore, a potential acquired function of mutant Htt is to intensify the effects of extracellular signals that function thorough the IKK complex.
A novel aspect of the present data is the mechanism by which mutant Htt stimulates NF-B. Mutant Htt activates the IKK complex both in cell culture and in mouse models of HD (Fig. 3). Our evidence indicates that activation of the IKK complex is likely caused by binding of mutant HDx1 to IKK. Optimal HDx1 binding requires interaction with the first N-terminal 134 amino acids of IKK (Fig. 4), a domain that is essential for interaction with IKK and IKK, two catalytic units of the IKK complex (May et al., 2000). It is plausible that mutant Htt binding to this region of IKK triggers oligomerization, a step that is essential for signal-induced IKK activation (Poyet et al., 2000; Tegethoff et al., 2003). It cannot be ruled out, however, that Htt-IKK binding mediates IKK interaction with other regulators responsible for phosphorylation and activation of the complex (Ghosh and Karin, 2002). Interaction of Htt with IKK requires polyQ expansion as well as the proline-rich motifs of HDx1. A similar scenario has been reported for the interaction of CBP and several WW-rich motif proteins implicated in HD pathology (Faber et al., 1998; Steffan et al., 2001). We find that recombinant intrabodies targeted to the polyP or polyQ motifs can inhibit Htt-IKK binding and NF-B-mediated gene expression. The anti-polyP intrabody, which is more potent, also reduces mutant HDx1 aggregation and protects cells from the toxicity of mutant Htt (Khoshnan et al., 2002). Therefore, the toxicity of mutant HDx1 may be reduced by interference with its binding to the IKK complex.
Expression of IKK also promotes aggregation and nuclear localization of mutant HDx1 before the widespread onset of toxicity in cell culture. Considering the propensity of both mutant HDx1 and IKK to oligomerize, their interaction may lead to enhanced aggregation of both proteins. Although the role of macro-inclusions in HD pathology is controversial, nuclear localization is thought to be a determinant of pathology (Saudou et al., 1998). IKK, which enters the nucleus (Anest et al., 2003; Yamamoto et al., 2003), is associated with nuclear HDx1 aggregates (Fig. 5A). Thus, IKK-induced nuclear localization of mutant HDx1 may influence Htt function. Conversely, binding of mutant Htt to the IKK complex may regulate nuclear IKK function.
The inhibition of mutant HDx1 toxicity by DN-IKK in cell culture and in MSNs is likely attributable to deregulation of IKK and subsequent inhibition of NF-B activity. We cannot rule out the possibility, however, that DN-IKK inhibits mutant HDx1 toxicity by an as yet unknown mechanism. Because both IKK and mutant Htt interact with CBP and regulate CBP-mediated gene expression (Nucifora et al., 2001; Verma et al., 2004), binding of IKK to mutant Htt could influence CBP sequestration and Htt toxicity; however, the similar effects of DN-IKK and F-TrCP in blocking cell death in cell culture and in MSNs support a role for NF-B in mutant Htt toxicity. Because IKK activity and TrCP are also essential for processing of the NF-B precursors p100 and p105 to the p52 and p50 subunits, respectively, DN-IKK and F-TrCP may also interfere with the generation of active NF-B complexes and reduce the available NF-B pool (Xiao et al., 2001; Fong and Sun, 2002).
Mutant Htt-induced activation of IKK can influence other signaling pathways that intersect with the NF-B pathway. For instance, Akt, a modulator of IKK activity and a substrate for NF-B, regulates the toxicity of mutant Htt and ataxin-1 (Humbert et al., 2002; Meng et al., 2002; Chen et al., 2003; Emamian et al., 2003). In addition, the wingless and NF-B pathways share the E3-TrCP ubiquitin ligase, which is essential for degradation of both IB and -catenin (Winston et al., 1999). Thus, the inhibition of mutant HDx1-induced toxicity by F-TrCP may also be mediated by a reduction in -catenin turnover. Mutant Htt can either stabilize or promote degradation of -catenin (Carmichael et al., 2002; Gines et al., 2003). Mutant Htt also induces expression of cyclin D1, which is downstream of -catenin (Gines et al., 2003), whereas signal-induced IKK phosphorylation of -catenin promotes cyclin D1 expression (Albanese et al., 2003). Thus, the interaction between mutant Htt and the IKK complex can influence a number of signaling pathways and represents an interesting target for intervention in polyglutamine-induced toxicity.
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Footnotes
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Received March 25, 2004;
revised July 30, 2004;
accepted July 31, 2004.
Support for this project was provided by the Hereditary Disease Foundation and by a grant (NS045165-01A1) from the National Institute of Neurological Disorders and Stroke awarded to P.H.P and A. K. We thank E. Schweitzer for providing ecdysone inducible PC12 cells, M. McDonald and S. Gines for mouse striatal cells, W. Greene for the GST-IB construct, E. Zandi for IKK plasmids, R. Deshaies for F-TrCP, E. Wanker for GST-HDx1, D. Housman and A. Kazantsev for HDx1-103Q-EGFP, B. Kerr for dissection of brain tissue from HD mice, M. Henson, D. Dunn, and D. Lo for helpful discussions and work with the brain slice experiments, and D. McDowell for administrative assistance.
Correspondence should be addressed to Ali Khoshnan, Biology Division 216-76, California Institute of Technology, Pasadena, CA 91125. E-mail: khoshnan{at}caltech.edu.
Copyright © 2004 Society for Neuroscience 0270-6474/04/247999-10$15.00/0
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Abstract
Introduction
