Expression of Expanded Polyglutamine Proteins Suppresses the Activation of Transcription Factor NFκB*

A major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones, and proteasome components. However, how the expanded polyglutamine proteins or their aggregates elicit complex pathogenic responses in the neuronal cells is not fully understood. Here, we have demonstrated that the expression of expanded polyglutamine proteins down-regulated the NFκB-dependent transcriptional activity. The expression of expanded polyglutamine proteins increased the stability and the levels of IκB-α and its phosphorylated derivatives. We have also found that various NFκB subunits and IκB-α aberrantly interacted with the expanded polyglutamine proteins and associated with their aggregates. Finally, we have shown that several NFκB-dependent genes are down-regulated in the expanded polyglutamine protein-expressing cells and down-regulation of NFκB activity enhances expanded polyglutamine protein-induced cell death. Because the NFκB pathway plays a very important role in cell survival, altered regulation of this pathway in expanded polyglutamine protein-expressing cells might be linked with the disease pathogenesis.

A major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones, and proteasome components. However, how the expanded polyglutamine proteins or their aggregates elicit complex pathogenic responses in the neuronal cells is not fully understood. Here, we have demonstrated that the expression of expanded polyglutamine proteins downregulated the NFB-dependent transcriptional activity. The expression of expanded polyglutamine proteins increased the stability and the levels of IB-␣ and its phosphorylated derivatives. We have also found that various NFB subunits and IB-␣ aberrantly interacted with the expanded polyglutamine proteins and associated with their aggregates. Finally, we have shown that several NFB-dependent genes are down-regulated in the expanded polyglutamine protein-expressing cells and down-regulation of NFB activity enhances expanded polyglutamine protein-induced cell death. Because the NFB pathway plays a very important role in cell survival, altered regulation of this pathway in expanded polyglutamine protein-expressing cells might be linked with the disease pathogenesis.
A common pathological feature of most age-related neurodegenerative disorders, including polyglutamine diseases, is the accumulation of intracellular protein deposits as inclusion bodies. Polyglutamine diseases are a group of familial neurodegenerative disorders that are caused by an abnormal expansion of CAG triplet repeats in the coding region of the target gene. These include Huntington disease (HD), 3 dentatorubral pallidoluysian atrophy, X-linked spinal bulbar muscular atrophy, and several spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17). All of these disorders are dominantly inherited (except X-linked spinal bulbar muscular atrophy), progres-sive, usually begin in midlife, and result in severe neuronal dysfunction and neuronal cell death in the selective region of the brain (1)(2)(3)(4).
NFB is a ubiquitous transcription factor that regulates the transcription of many genes involved in immune and inflammatory responses as well as cell survival and cell death (21)(22)(23). NFB is localized in the cytoplasm in an inactive form in association with a family of inhibitory proteins (IBs). In response to multiple activating signals, IB is phosphorylated and subsequently degraded by the UPS. The rapid degradation of IB proteins unmasks the nuclear localization signals of NFB, which then translocate to the nucleus and activate the transcription of multiple genes. The activation of NFB seems to stimulate some pathways that promote cell death, whereas other pathways promote cell survival (22,23).
Several neurotrophic factors, cytokines, and excitotoxic neurotransmitters have been found to activate the NFB pathway, and in most cases, activation leads to neuroprotection (22, 24 -26). The NFB activation in neurons is also observed in acute neurodegenerative conditions such as stroke and ischemic brain injury (22,27,28) and in chronic neurodegenerative disorders such as Alzheimer disease, HD, and amyotrophic lateral sclerosis (29 -33). The increased NFB activity in the affected neurons might be an early protective response to ongoing oxidative stress or mitochondrial dysfunction (34). This is supported by the fact that the mice lacking the p50 subunit of NFB shows increased damage of striatal neurons and motor disturbances after administration of mitochondrial toxin 3-nitropropionic acid (31).
We had reported earlier the impairment of UPS function in the expanded polyglutamine protein-expressing cells, which might be linked with cell death. But how the UPS dysfunction leads to the death of expanded polyglutamine protein-express-ing cells is not well understood. Because the NFB activation is essential for cell survival and is highly regulated by the UPS (35), we investigated the possible role of the NFB pathway in the expanded polyglutamine protein-induced cell death. Here, we report that the expression of expanded polyglutamine proteins causes down-regulation of NFB activity and promotes expanded polyglutamine protein-induced cell death.
Cell Culture, Transfection, Cell Viability, and Reporter Gene Assay-The wild-type mouse neuro-2a and COS-1 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and antibiotics penicillin/streptomycin. The stable neuro-2a cell lines HD 16Q and HD 150Q were maintained in the same medium containing 0.4 mg/ml zeocin and 0.4 mg/ml G418. One day prior to transfection, the cells were plated into 6-well tissue-cultured plates at a subconfluent density. For the reporter gene assay, cells were transiently transfected with NFB-luciferase and PRL-SV40 plasmids together using Lipofectamine 2000 reagent according to the manufacturer's instructions. Transfection efficiency was ϳ80 -90%. The cells were left untreated or induced with ponasterone A (1 M) for different time periods and then processed for luciferase assay. In some experiments, cells were induced for 2 days, treated with different NFB-modulating agents for 8 h, and then processed for luciferase assay. In another experiment, the neuro-2a cells were first transfected with pEGFP-N1-MJD(t)-20CAG and pEGFP-N1-MJD(t)-130CAG plasmids followed by NFB-luciferase and PRL-SV40 plasmids. Luciferase activity was measured using the dual luciferase reporter assay system according to the manufacturer's instructions. PRL-SV40 was used for co-transfection to normalize the data and transfected at a very low concentration (150-fold lower than NFB luciferase plasmid). Data were represented as relative luciferase activity (the ratio of firefly to Renilla values). For the cell viability assay, HD 16Q and HD 150Q cells were plated into 96-well plates (5 ϫ 10 3 cells/well). The cells were then induced with 1 M ponasterone A for 2 days and then treated with various agents for 8 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described previously (37). The proteasome assay was performed as described earlier (38,39). Statistical analysis was performed using Student's t test, and p Ͻ 0.05 was considered to indicate statistical significance.
Co-immunoprecipitation and Immunoblotting Experiment-After 24 or 48 h of induction, cells were washed with cold phosphate-buffered saline, scraped, pelleted by centrifugation, and lysed on ice for 30 min with Nonidet P-40 lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, complete protease inhibitor mixture). Cell lysates were briefly sonicated, centrifuged for 10 min at 15,000 ϫ g at 4°C, and the supernatants (total soluble extract) were used for immunoprecipitation as described earlier (38,39). For each immunoprecipitation experiment, 200 g of protein in 0.2 ml of Nonidet P-40 lysis buffer was incubated either with 5 l (2 g) of GFP antibody or 4 l (2 g) of normal mouse IgG. Bound proteins were eluted from the beads with SDS (1ϫ) sample buffer, vortexed, boiled for 5 min, and analyzed by immunoblotting according to the procedure described earlier (38,39). Blot detection was carried out with enhanced chemiluminescence reagent. All primary antibodies were used at 1:1000 dilutions for immunoblotting.
Immunofluorescence Techniques-Cells grown in chamber slides were induced with ponasterone A. Forty-eight hours after induction, the cells were washed twice with phosphate-buffered saline, fixed with 4% paraformaldehyde in phosphate-buffered saline for 20 min, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 min, washed extensively, and then blocked with 5% nonfat dried milk in TBST (Tris-buffered saline with Tween 20) for 1 h. Primary antibody (anti-NFB and anti-IB-␣, 1:500 dilutions; anti-ataxin-3, 1:1000 dilutions) incubation was carried out overnight at 4°C. After several washings with TBST, the cells were incubated with rhodamineconjugated secondary antibody (1:500 dilutions) for 1 h, washed several times, and mounted in anti-fade solution. The samples were observed using a confocal microscope, and digital images were assembled using Adobe Photoshop.
Degradation Assay-HD 16Q and HD 150Q cells were plated in a 6-well tissue-cultured plate and induced with ponasterone A for 48 h. The cells were chased with 10 g/ml of cycloheximide for different time periods. The cells collected at each time point were then processed for immunoblotting using antibodies against IB-␣ and NFB p65 and p50 subunits.

The Levels of IB-␣ and Its Phosphorylated Derivatives Is Increased in Expanded Polyglutamine Protein-expressing Cells-
We developed several stable neuro-2a cell lines in an inducible system that expresses tNhtt with normal (16Q) and expanded polyglutamine (150Q) proteins (37). These cell lines were named HD 16Q and HD 150Q, and their corresponding expressed proteins were named tNhtt-16Q and tNhtt-150Q. The cell lines were induced for different time periods with ponasterone A (1 M) and then processed for immunoblotting using antibodies against IB-␣, p-IB-␣, NFB p65 and p50 subunits, and ␤-tubulin. As shown in Fig.  1A, expression of tNhtt-150Q protein time-dependently increased the levels of IB-␣ and p-IB-␣. There was no change of the levels of IB-␣ and slight increase in p-IB-␣ in the HD 16Q cells upon expression of tNhtt-16Q protein. The levels of IB-␣ and its phosphorylated derivatives in the uninduced HD 150Q cells were much higher compared with uninduced HD 16Q cells. This is most likely because of the low levels of expression and aggregation of tNhtt-150Q protein without any induction (37). The levels of NFB p65 and p50 subunit were not changed in both HD 16Q and HD 150Q cells upon different days of induction.
We had reported earlier the proteasomal dysfunction in the HD 150Q cells (15). Therefore, we presumed that the increased accumulations of IB-␣ and its phosphorylated derivatives might be due to the altered proteasomal function in these cells. To confirm this hypothesis, we determined the proteasome activity in HD 16Q and HD 150Q cells upon different days of induction. As shown in Fig. 1B, the expression of expanded polyglutamine proteins time-dependently reduced the proteasome activity in HD 150Q cells. This inhibition of proteasome activity correlated well with the increased accumulation of IB-␣ and its phosphorylated derivatives. The slight increase in p-IB-␣ in the HD 16Q cells also might be due to the proteasomal dysfunction at higher levels of expression of tNhtt-16Q proteins. The HD 150 cells also show changes in morphology upon induction compared with HD 16Q cells. The cells exhibit neurite outgrowth and look bipolar. This again might be due to proteasomal dysfunction, because proteasome inhibitors are shown to induce neurite outgrowth.
Expression of Expanded Polyglutamine Proteins Increases the Stability of IB-␣-Because the levels of IB-␣ and its phosphorylated forms are increased in HD 150Q cells upon induction, we assumed altered degradation of IB-␣ in these cells. Therefore, we checked the half-life of IB-␣ in both HD 16Q and HD 150Q cells after induction with ponasterone A for 2 days fol-  lowed by cycloheximide chase. More than 50% of HD 150Q cells form aggregates at 2 days of induction. Fig. 2 shows that the half-life of IB-␣ dramatically increased in the HD 150Q cells in comparison with HD 16Q cells. The half-life of NFB p65 and p50 subunits were not affected in both HD 16Q and HD 150Q cells when they were induced for 2 days and performed chase experiments.
Association of IB-␣ and NFB p65 with Polyglutamine Aggregates-Next, we searched for clues of increased half-life of IB-␣ and their consequences in the HD 150Q cells. One of the reasons for increased half-life of IB-␣ in the HD 150Q cells could be due to UPS dysfunction, because we have reported the inhibition of UPS function in these cells after 2 days of induction (15). The increased accumulation of IB-␣ might lead to the inhibition of NFB activity by preventing their nuclear translocation. To confirm this fact, we first performed immunofluorescence staining of the NFB p65 subunit in the wild-type neuro-2a cells as well as in the HD 16Q and HD 150Q cells after 2 days of induction. The NFB p65 protein was mostly localized in the cytoplasm with weak nuclear staining in the wild-type neuro-2a cells (data not shown). The expression of tNhtt-16Q in HD 16Q cells did not alter the distribution profile of NFB p65 protein, in comparison with wild-type neuro-2a cells (Fig.  3). NFB p65 was mostly localized in the cytoplasm in HD 16Q cells. However, to our surprise, we noticed that the expression of tNhtt-150Q protein in HD 150Q cells caused recruitment of NFB p65 protein to the mutant huntingtin aggregates. We also observed reduction of the diffuse nuclear staining of NFB p65 protein in these cells. Next, we performed immunofluorescence staining of IB-␣ in the wild-type neuro-2a, HD 16Q, and HD 150Q cells. The HD 16Q and HD 150Q cells were similarly induced for 2 days. The IB-␣ was normally distributed in both the cytoplasmic and nuclear compartments, with predominant nuclear localization in the wild-type neuro-2a and uninduced HD 16Q cells. The expression of tNhtt-16Q protein in HD 16Q cells did not alter the distribution pattern of IB-␣, as compared with uninduced HD 16Q cells (Fig. 4). The expression of tNhtt-150Q in HD 150Q cells caused recruitment of IB-␣ to the mutant huntingtin aggregates, similar to NFB p65 protein (Fig. 4). It seems that the whole NFB complex is sequestered around the aggregates. Next, we performed co-immunoprecipitation experiments to confirm the interaction of soluble expanded polyglutamine proteins with the NFB p65 and IB-␣. The HD 16Q and HD 150Q cell lines were induced for 1 day with ponasterone A (1 M) and then processed for immunoprecipitation by anti-GFP. In other experiments, we transfected the truncated ataxin-3 constructs (pEGFP-N1-MJD(t)-20CAG and pEGFP-N1-MJD(t)-130CAG) to the COS-1 cell, and after 1 day, the cells were collected and processed for immunoprecipitation by the GFP antibody. Blots were probed with anti-IB-␣ and anti-NFB p65. Fig. 5 shows that both IB-␣ and NFB p65 are specifically interacted with polyglu-  tamine-expanded huntingtin or ataxin-3. The tNhtt-150Q or ataxin-3(t)-130Q appeared as multiple bands because of the instability of the CAG repeats.
To further confirm the association of the NFB complex with expanded polyglutamine proteins or their aggregates and to exclude any interference of GFP, we transiently transfected truncated ataxin-3 constructs without the GFP tag. At 48 h of post-transfection, the cells were processed for either immunofluorescence or immunoblotting experiments using IB-␣, NFB p65, and ataxin-3 antibodies. As expected, both IB-␣ and NFB p65 proteins were interacted and associated with the polyglutamine-expanded ataxin-3 (Fig. 6).

Down-regulation of NFB-dependent Transcriptional Activity in the Expanded Polyglutamine Protein-expressing Cells-
Because the half-life of IB-␣ is increased in HD 150Q cells and the NFB complex sequestered around the aggregates, we assumed that there might be down-regulation of NFB activity in the HD 150Q cells. As expected, the expression of tNhtt-150Q protein in HD 150Q cells time-dependently decreased the NFB-dependent transcriptional activity (Fig. 7A). The expression of tNhtt-16Q protein in HD 16Q cells did not have any effect on the NFB-dependent transcriptional activity. The NFB-dependent transcriptional activity was significantly lower in the uninduced HD 150Q cells as compared with HD 16Q cells most likely because of the leaky expression and aggregation of tNhtt-150Q proteins. Next, we determined the NFBdependent transcriptional activity in both HD 16Q and HD 150Q cells after treating with various modulators of the NFB pathway. We used lipopolysaccharide as a stimulator and curcumin and MG132 as inhibitors of NFB activation. Fig. 7B shows that treatment of lipopolysaccharide increased NFB activity in HD 16Q cells while preventing the NFB activation in HD 150Q cells. The curcumin and MG132 both enhanced the down-regulation of NFB activity in the HD 150Q cells. We further checked the NFB activity in the neuro-2a cells after transiently expressing the ataxin-3(t)-20Q and ataxin-3(t)-130Q proteins. In this case, we observed activation of NFB in polyglutamine-expanded ataxin-3-expressing cells at 24 h of post-transfection, whereas we observed inhibition after 72 h of post-transfection (Fig. 7C). Possibly, the soluble expanded polyglutamine proteins are initially activating the NFB, whereas later polyglutamine aggregates inhibit the NFB activity.
We next checked the expression levels of several NFB-dependent genes in the HD 16Q and HD 150Q cells after different days of induction. As shown in figure 8, the expression of tNhtt-150Q proteins in the HD 150Q cells results in down-regulation of Cox2 and iNOS expression. The expression levels of Cox2 and iNOS were very low in the uninduced HD 150Q cells. This was expected, because the uninduced HD 150Q cells showed a significantly low level of NFB activity as compared with HD 16Q cell.
Inhibition of NFB Enhances the Expanded Polyglutamine Protein-induced Cell Death-Because the NFB activity was inhibited in the expanded polyglutamine protein-expressing cells, we next checked the impact of this down-regulation on cell viability. We induced the HD 16Q and HD 150Q cells for 2 days with ponasterone A and then treated them with varying doses of SN50 (a peptide inhibitor of NFB that blocks nuclear import of the NFB p50 subunit protein) or curcumin. Curcumin is well known to inhibit NFB activity (40). We induced HD 150Q cells for 2 days, because there was significant reduction of NFB activity, and these cells start dying from 2 days onward (15). Fig. 9 shows that treatment of both SN50 and curcumin induced HD 16Q and HD 150Q cell death. However, the percentage of HD 150Q cell death was significantly higher in comparison with HD 16Q cells. Results suggest that the NFB inhibitors promote expanded polyglutamine protein-induced cell death.

DISCUSSION
Activation of NFB plays a very important role in controlling the pathways related to cell survival and apoptosis. Activation of this pathway is also implicated in various acute and chronic neurodegenerative conditions. In the present investigation, we have found that the expression of expanded polyglutamine proteins time-dependently down-regulates the NFB activity.
First, we have shown that the expression of expanded polyglutamine protein results in an increased accumulation of IB-␣ and its phosphorylated derivatives, and this increased accumulation is because of the slower rate of degradation of IB-␣. Second, we found that the IB-␣ and NFB subunits aberrantly interact with the expanded polyglutamine proteins and sequester around the aggregates. Therefore, most likely the association of IB-␣ and NFB complexes with the polyglutamine aggregates, as well as altered degradation of IB-␣, might be responsible for the down-regulation of NFB activity.
Polyglutamine aggregates have been reported to associate with the various components of UPS (15)(16)(17)(18)(19)(20), and continuous expression and aggregation of expanded polyglutamine proteins in neuronal cells lead to the impairment of UPS function (12)(13)(14)(15). Therefore, the altered degradation of IB-␣ could be due to the proteasomal dysfunction in the expanded polyglutamine protein-expressing cells. Aberrant association of IB-␣  with the polyglutamine aggregates could also increase its half-life.
Expanded polyglutamine proteins are very unstable and quickly form aggregates (41), and during this process, they coaggregate with many other proteins essential for normal cellular function. Among them, several transcription factors, such as TATA box-binding protein, CREB-binding protein, Sp1, and p53 have been found to associate with aggregates and result in their transcriptional deregulation, which ultimately leads to cellular dysfunction and cell death (5)(6)(7)(8)(9)(10)(11). Here, we found transcriptional deregulation of NFB in the expanded polyglutamine protein-expressing cells, which might be due to the UPS dysfunction as well as sequestration of various components of NFB complex and could be a common feature among all polyglutamine diseases. Finally, we have also shown that some of the NFB-regulated genes, such as Cox2 and iNOS, are down-regulated in the expanded polyglutamine protein-expressing cells.
What could be the consequence of the down-regulation of NFB activity in the expanded polyglutamine protein-expressing cells? NFB activation has been reported to protect as well as to promote cell death, depending on the cell type and/or inducing signal (22)(23)(24). But in most cases, its activation leads to the protection of cell death, particularly in the neuronal cells (22). NFB activity was reported to be increased in the striatal neurons of 3-nitropropionic acid-treated mice (an animal model for HD). However, the mice lacking the p50 subunit of NFB show decreased activity and increased damage of striatal neurons (31). This suggests that down-regulation of NFB activity might lead to neurodegeneration. However, a recent paper shows activation of NFB in the cellular and transgenic mice model of HD, and this activation leads to neurodegeneration (32). We never detected an increase in NFB activity in HD 150Q stable cells most likely because of a low level of expression and aggregation of polyglutamine-expanded huntingtin without any induction. However, we have found a significant increase in NFB activity at 24 h of post-transfection and a decrease in NFB activity at 72 h of post-transfection of the polyglutamine-expanded ataxin-3. In both cases, we have observed direct correlation of decrease in NFB activity and induction of cell death. It seems that expanded polyglutamine protein could possibly have a dual effect on NFB, an initial phase of activation and a later stage of inhibition. Initially, the expanded polyglutamine protein-expressing cells might be trying to protect themselves by activating NFB, and later aggregates prevent the activation by promoting UPS dysfunction. Similarly, the amyloid ␤-peptide at low concentration has been found to activate NFB that leads to neuroprotection, whereas at a higher concentration, it produces the opposite effect (29). We have observed down-regulation of NFB activity promoting expanded polyglutamine protein-induced cell death, which suggests that inhibition of NFB activity might be responsible for the expanded polyglutamine protein-induced cell death. Further work is necessary to confirm this.
Altogether, our results demonstrate the down-regulation of NFB activity in the expanded polyglutamine protein-expressing cell, and this inhibition of NFB activity might be linked with the expanded polyglutamine protein-induced cell death.