TDP-43 interacts with the p65 subunit of NF-κB
Mass spectrometry analysis and coimmunoprecipitation experiments were performed to identify proteins that interact with TDP-43 in mouse microglia (BV-2) cells after LPS stimulation, as described in Materials and methods. Many proteins were coimmunoprecipitated with TDP-43, including proteins responsible for RNA granule transport (kinesin), molecular chaperones (Hsp70), and cytoskeletal proteins (unpublished data). In addition, our analysis revealed p65 (REL-A
) as a novel protein interacting with TDP-43. An interaction between TDP-43 with p65 NF-κB was confirmed by a coimmunoprecipitation assay with a polyclonal antibody against TDP-43 using spinal cord extracts from transgenic mice overexpressing human TDP-43WT
mutant (Swarup et al., 2011
) by threefold (). Additional coimmunoprecipitation experiments performed using BV-2 cells that were transiently transfected with pCMV–TD-P43WT
and pCMV-p65 plasmids clearly showed that TDP-43 interacts with p65.
Figure 1. TDP-43 interacts with NF-κB p65. (A) Protein extracts from the spinal cords of nine sporadic ALS subjects (1–9) and six control individuals (1–6) were used for the immunoprecipitation (IP) with TDP-43–specific polyclonal (more ...)
To further determine the significance of TDP-43 interaction with p65 in the context of human ALS, TDP-43 was pulled down with the polyclonal anti–TDP-43 antibody using spinal cord extracts from nine sporadic ALS cases and six control subjects (). In protein extracts from ALS cases, p65 NF-κB was coimmunoprecipitated with TDP-43. In contrast, no p65 was pulled down with TDP-43 using extracts of control spinal cords. To further validate TDP-43–p65 interaction, we performed reverse coimmunoprecipitation using p65 antibody to immunoprecipitate TDP-43 in human spinal cord tissues. Indeed, p65 was able to coimmunoprecipitate TDP-43 in all nine ALS cases but not in six control cases (Fig. S1 A
). Along with p65, p50 was also coimmunoprecipitated with TDP-43 from the spinal cord samples of TDP-43WT
mice and ALS samples but not from nontransgenic or control spinal cord tissues, suggesting that TDP-43, p50, and p65 are a part of a complex (). To determine whether TDP-43 interacts directly with p65 or p50, we have performed overexpression experiments using pCMV expression vectors transfected into mouse neuroblastoma Neuro2a cells (). Neuro2a cells were transfected with pCMV-p65 or pCMV-p50 expression vectors along with vectors encoding either hemagglutinin (HA)-tagged TDP-43WT
, a deletion mutant lacking the region required for binding to p65 as described in the section p65 interacts with the N-terminal and RRM-1 domains of TDP-43. It should be noted that the cells were not stimulated by LPS or any other means. After overexpression of p65 and TDP-43WT
in the Neuro2a cells, p65 was coimmunoprecipitated with TDP-43WT
but not with TDP-43ΔNR1-30
using anti-HA antibody. In contrast, p50 was not coimmunoprecipitated with TDP-43WT
when overexpressed alone with TDP-43. These results suggest that TDP-43 interacts directly with p65 but not directly with p50. Immunofluorescence microscopy corroborated these results. In the spinal cord of sporadic ALS subjects, p65 was detected predominantly in the nucleus of cells in colocalization with TDP-43 (). On the contrary, in control spinal cord, there was an absence of p65 in the nucleus, reflecting a lack of p65 activation (). It is remarkable that microscopy of the spinal cord from TDP-43WT
transgenic mice revealed ALS-like immunofluorescence with active p65 that colocalized perfectly with TDP-43 in the nuclei of cells (). To elucidate which cell types in the spinal cord of ALS cases express TDP-43 and p65, we performed three-color immunofluorescence with CD11b as a microglial-specific marker and glial fibrillary acidic protein (GFAP) as an astroglial marker. We found that TDP-43 and p65 colocalized in many microglial and astroglial cells (, insets). We have quantified our data and found that 20 ± 5% of microglia and 8 ± 3% of astrocytes had TDP-43–p65 colocalization. We also found that many of the TDP-43 p65 colocalization was in neurons, and some also in motor neurons in many ALS cases (). In many ALS cases in which TDP-43 formed aggregates in the cytoplasm, p65 was still in the nucleus (, arrowheads). In nontransgenic C57BL/6 mice, the lack of p65 activation resulted in partial colocalization of TDP-43 with p65 mainly in cytoplasm (). LPS-stimulated BV-2 cells transfected with pCMV-p65 and pCMV–TDP-43WT
had most p65 colocalized with nuclear TDP-43WT
, whereas in unstimulated cells, p65 did not colocalize with nuclear TDP-43WT
. Although p65 was mainly cytoplasmic in 3-mo-old TDP-43WT
spinal cord, there was gradual age-dependent p65 activation in 6- and 10-mo-old TDP-43WT
spinal cord (Fig. S1 D).
Figure 2. TDP-43 colocalizes with p65 in neuronal and glial cells. (A–C) TDP-43 and p65 double immunofluorescence was performed in different sporadic ALS cases as indicated. Double immunofluorescence pictures were taken at various magnifications. Arrowheads (more ...)
TDP-43 acts as a co-activator of p65
A gene reporter assay was performed to study the effect of TDP-43 on NF-κB–dependent gene expression. The effect of TDP-43 was studied on gene expression of the reporter plasmid 4κBWT-luc by transfecting pCMV–TDP-43WT in BV-2 cells with or without cotransfection of pCMV-p65 (). When expressed alone, TDP-43 had no detectable effect on the basal transcription level of plasmid 4κBWT-luc, suggesting that TDP-43 does not alter the basal transcription level of NF-κB. However, in coexpression with p65, TDP-43 augmented the gene expression of plasmid 4κBWT-luc in a dose-dependent manner. 20 ng pCMV-p65 alone activated gene expression of 4κBWT-luc by 10-fold (). However, upon cotransfection with 20 ng pCMV–TDP-43WT, the extent of gene activation was elevated to 22-fold (2.2-fold augmentation by the effect of TDP-43). A further increase in NF-κB–dependent gene expression was recorded as the levels of TDP-43WT were elevated to 50 ng (2.8-fold activation) and 100 ng (3.2-fold activation; n = 4; P < 0.05). When using a control luciferase reporter construct, 4κBmut-luc, in which all four κB sites were mutated, neither the activation by pCMV-p65 nor the effect of cotransfection of pCMV–TDP-43WT was detected. The boosting effects of TDP-43 were not caused by increased levels in p65 as shown by immunoblotting (). Similarly, pCMV–TDP-43A315T and pCMV–TDP-43G348C augmented p65-mediated gene expression from the reporter plasmid 4κBWT-luc (not depicted).
Figure 3. TDP-43 acts as a co-activator of NF-κB p65. (A) BV-2 cells were transfected with 20 ng 4κBWT-luc (containing WT NF-κB–binding sites) or 4κBmut-luc (containing mutated NF-κB–binding sites) together (more ...)
To further examine the effect of TDP-43 on the activation of p65, we performed p65 electrophoretic mobility shift assays (EMSAs). Transfection in BV-2 cells of pCMV-p65 with pCMV–TDP-43WT or pCMV–TDP-43G348C and LPS treatment was followed by extraction of nuclear proteins. Subsequently, the interaction between p65 in the protein extract and DNA probe was investigated using the EMSA kit from Panomics according to the manufacturer’s instructions. TDP-43 increased the binding of p65 to the NF-κB DNA probe in a dose-dependent manner. LPS alone induced the binding of p65 to the DNA probe by about twofold as compared with control (). The cotransfection of 50 and 100 ng TDP-43WT or of 100 ng TDP-43G348C resulted in a significant dose-dependent increase in the DNA binding of p65. The specificity of the gel shift assay was assessed by adding a cold probe. TDP-43 alone did not bind to p65 EMSA probes (Fig. S1 B). Moreover, adding an anti-HA antibody that recognizes the transfected TDP-43 or an anti-p65 antibody caused supershifts of bands in the p65 EMSA (). Along with p65 and TDP-43, p50 is also part of the activated complex as seen by supershifts of bands in p65 EMSA experiments in BV-2 cells using antibodies specific to p65, TDP-43, and p50 (Fig. S1 C).
p65 interacts with the N-terminal and RRM-1 domains of TDP-43
To determine which domains of TDP-43 interact with p65, we constructed a series of deletion mutants of various TDP-43 domains. Various pCMV-HA–tagged deletion mutants like TDP-43ΔN (1–105 aa), TDP-43ΔRRM-1 (106–176 aa), TDP-43ΔRRM-2 (191–262 aa), and TDP-43ΔC (274–414 aa) were transfected in BV-2 cells with pCMV-p65 (). TDP-43ΔRRM-1 coimmunoprecipitated p65 partially, whereas TDP-43ΔRRM-2 and TDP-43ΔC interacted well with p65, suggesting that RRM-1 is important but RRM-2 and C-terminal domains are dispensable for interaction with p65. After transfection, we found that TDP-43ΔN had much reduced interaction with p65 (), thereby suggesting that the N-terminal domain of TDP-43 is essential for the interaction of TDP-43 with p65. Because the nuclear localization signal is in the N terminus, the reduced interaction of TDP-43ΔN to p65 could have been the result of a mislocalization of TDP-43ΔN. To address this issue and to further define the interacting domain, we constructed a series of N-terminal and RRM-1 deletion mutants, TDP-43ΔNR1-81 (98–176 aa), TDP-43ΔNR1-50 (51–81 and 98–176 aa), and TDP-43ΔNR1-30 (31–81 and 98–176 aa), with the nuclear localization signal attached so that the mutant proteins are able to be directed to the nucleus. Coimmunoprecipitation with these constructs suggested that even though TDP-43ΔNR1-30 is in the nucleus (), it cannot effectively interact with p65, TDP-43ΔNR1-81, and TDP-43ΔNR1-50, whereas it can interact with p65 (). These results indicate that TDP-43 interacts with p65 through its N-terminal domain (31–81 and 98–106 aa) and RRM-1 (107–176 aa) domain.
Figure 4. The N-terminal and RRM-1 domains of TDP-43 are crucial for interaction with p65. (A) Two-dimensional cartoon of TDP-43 protein showing various deletion mutants used in this study. Deletion mutants TDP-43ΔN (1–105 aa), TDP-43ΔRRM-1 (more ...)
To assess the effect of these deletion mutants on the activation of NF-κB gene, we used the gene reporter assay. Various deletion mutants of TDP-43 were cotransfected along with 4κBWT-luc or 4κBmut-luc. When compared with full-length TDP-43WT, TDP-43ΔN had reduced effect (twofold; n = 3; P < 0.05) on the gene activation. TDP-43ΔRRM-1 also exhibited attenuation of gene activation but to a lesser extent than TDP-43ΔN (). In contrast, TDP-43ΔRRM-2 and TDP-43ΔC deletion mutants had effects similar to full-length TDP-43WT. As expected, because TDP-43ΔNR1-30 does not effectively interact with p65, the level of NF-κB activation detected by the 4κBWT-luc reporter assay was extremely low, sixfold lower than full-length TDP-43WT (n = 3; P < 0.001; ). p65 and luciferase vectors were used as controls for the experiment. Note that the amount of pCMV-p65 vector transfected in control was more than in other experiments to keep similar amounts of total transfected DNA. Transfection of a control luciferase reporter construct, 4κBmut-luc, in which all four κB sites were mutated, had no effect on the basal level activation of p65. To determine whether the interaction between TDP-43 and p65 is a protein–protein interaction, we performed immunoprecipitation experiments by adding proteinase K, RNase A, or DNase 1 (). The addition of proteinase K abolished the TDP-43–p65 interaction, whereas RNase A or DNase 1 had no effect, suggesting that the interaction is not DNA/RNA dependent.
TDP-43 small interfering RNA (siRNA) inhibits activation of NF-κB
If it is correct that TDP-43 acts as a co-activator of p65, then reducing the levels of TDP-43 should attenuate p65 activation. To reduce the expression levels of TDP-43, microglial BV-2 cells were transfected with either TDP-43 siRNA or scrambled siRNA together with 4κBWT-luc vectors. 72 h after transfection, some of the cells were either stimulated with 100 ng/ml LPS or mock stimulated for 12 h. As shown in , TDP-43 siRNA reduced the endogenous mouse TDP-43 levels significantly as compared with scrambled siRNA–transfected cells in two different experiments. To examine the effect of reducing TDP-43 levels on NF-κB activation, BV-2 cells were transfected with pCMV-p65 and 4κBWT-luc vectors. TDP-43 siRNA decreased activation of NF-κB reporter gene in transfected cells. The decrease in NF-κB activation was about threefold for 5 ng pCMV-p65 (n = 4; P < 0.01), ~2.5-fold for 10 and 20 ng pCMV-p65 (n = 4; P < 0.05), and twofold for 50 ng pCMV-p65 (n = 4; P < 0.05) as compared with scrambled siRNA–transfected cells (). To examine the physiological significance of TDP-43 inhibition by siRNA, we transfected BV-2 cells with ICAM-1–luc vector together with TDP-43 siRNA or scrambled siRNA. 72 h after transfection, cells were stimulated with varying concentrations of TNF. When stimulated with 0.5 ng/ml TNF, TDP-43 siRNA–transfected cells exhibited a twofold decrease in ICAM-1 luciferase activity (n = 4; P < 0.05) as compared with cells transfected with scrambled siRNA. Similarly, TDP-43 siRNA–transfected BV-2 cells exhibited at 1.0- and 1.5-ng/ml TNF concentrations a decrease of 2.5-fold (n = 4; P < 0.01) and twofold (n = 4; P < 0.05) in ICAM-1 luciferase activity, respectively (). We also tested the effect of TDP-43 siRNA transfected in BM-derived macrophages (BMMs) from normal mice. We compared the level of innate immunity activation when stimulated with LPS. BMMs transfected with TDP-43 siRNA had reduced levels of TLR2 mRNA (1.5-fold; P < 0.05), p65 (threefold; P < 0.01), TNF (threefold; P < 0.01), IL-1β (twofold; P < 0.05), IP-10 (twofold; P < 0.05), IL-6 (2.5-fold; P < 0.01), and Cox-2 (cyclooxygenase-2; twofold; P < 0.05) as compared with scrambled siRNA–transfected BMMs ().
Figure 5. TDP-43 siRNA inhibits activation of NF-κB. BV-2 cells were transfected either with mouse TDP-43 siRNA or scrambled siRNA. 72 h after transfection, some of the cells were either stimulated with 100 ng/ml LPS or mock stimulated for 12 h. (A) Protein (more ...)
TDP-43 and p65 mRNA levels are up-regulated in the spinal cord of sporadic ALS patients
The findings that TDP-43 can interact with p65 and that TDP-43 overexpression in transgenic mice was sufficient to provoke abnormal nuclear colocalization of p65 as observed in sporadic ALS () prompted us to compare the levels of mRNA coding for TDP-43 and p65 NF-κB in spinal cord samples from sporadic ALS cases and control individuals. Real-time RT-PCR data revealed that the levels of TDP-43 mRNA in the spinal cord of sporadic ALS cases (n = 16) were up-regulated by ~2.5-fold (P < 0.01) compared with controls (n = 6; ). It is also noteworthy that the levels of p65 NF-κB mRNA were up-regulated by about fourfold (P < 0.001) in ALS cases as compared with controls. Because TDP-43 forms many bands in Western blot analysis, we quantified the total level of TDP-43 protein using sandwich ELISA as described in Materials and methods. The ELISA results suggest that TDP-43 protein levels are in fact up-regulated in total spinal cord protein extracts of ALS cases (n = 16) by 1.82-fold (241.2 ± 8.5 pg/µg of total protein) as compared with control cases (132.8 ± 5.6 pg/µg of total protein; n = 6; ). For human p65 ELISA, we used an ELISA kit from QIAGEN. The levels of p65 were also up-regulated in total spinal cord extracts of ALS cases (n = 16) by 3.5-fold (222.5 ± 11.5 pg/µg of total protein) as compared with control cases (62.83 ± 3.8 pg/µg of total protein; n = 6; ).
Figure 6. Analysis of TDP-43 and NF-κB p65 mRNA expression in sporadic ALS spinal cord. (A) Spinal cord tissue samples from 16 different sporadic ALS patients and 6 controls were subjected to real-time RT-PCR analysis using primers specific for TDP-43 (TARDBP) (more ...)
TDP-43 overexpression in glia or macrophages causes hyperactive inflammatory responses to LPS
Because NF-κB is involved in proinflammatory and innate immunity response, we tested the effects of increasing TDP-43 mRNA expression in BV-2 cells. Because LPS is a strong proinflammatory stimulator (Horvath et al., 2008
), we used it to determine the differences in levels of proinflammatory cytokines produced by TDP-43–transfected or mock-transfected BV-2 cells. BV-2 cells were transiently transfected with pCMV–TDP-43WT
, or empty vector. 48 h after transfection and 12 h after 100-ng/ml LPS challenge, RNA extracted from various samples was subjected to real-time quantitative RT-PCR to determine the mRNA levels of various proinflammatory genes. As expected, there was a fourfold increase in mRNA levels of TNF after LPS stimulation of BV-2 cells compared with controls (). However, in LPS-treated cells transfected with WT TDP-43, there was an additional threefold (n
= 5; P < 0.05) increase in TNF levels. TDP-43 harboring the A315T and G348C mutations had similar effects on boosting the levels of TNF upon LPS stimulation. Similarly, in response to LPS, the extra levels of TDP-43 species in transfected microglial cells caused a significant fivefold increase (n
= 5; P < 0.001) in the mRNA levels of IL-1β () and ninefold increase in mRNA levels of IL-6 (n
= 5; P < 0.001; ) as compared with LPS-treated mock-transfected cells. The levels of NADPH oxidase 2 (Nox-2 gene) was increased by ~2.8-fold (n
= 5; P < 0.05; ) in LPS-challenged TDP-43–transfected cells as compared with LPS-treated mock-transfected cells. Remarkably, overexpression of TDP-43 species resulted in a 10-fold (n
= 5; P < 0.001) increase in levels of p65 (RELA) mRNA in LPS-treated transfected cells as compared with LPS-treated mock-transfected cells (). Note that, in the absence of LPS stimulation, microglial cells transfected with TDP-43 species (both WT and mutants) exhibited no significant differences in levels of TNF, IL-1β, Nox-2, and NF-κB when compared with mock-transfected controls.
Figure 7. Analysis of genes involved in inflammation of mouse microglial and macrophage cells overexpressing human TDP-43. (A–C) Mouse microglial cells BV-2 were either transfected with pCMV–TDP-43WT, pCMV–TDP-43A315T, and pCMV–TDP-43 (more ...)
Figure 8. TDP-43 up-regulation enhances neuronal vulnerability to death by microglia-mediated cytotoxicity. (A and B) TDP-43 (WT and mutants)–transfected BV-2 cells were stimulated with LPS. 12 h after stimulation, total RNA was extracted with TRIZOL. The (more ...)
To further evaluate the effect of LPS stimulation in TDP-43–overexpressing microglia, we prepared primary microglial cultures from C57BL/6 mice and from transgenic mice overexpressing TDP-43WT by threefold. Primary microglial cells were challenged with LPS at a concentration of 100 ng/ml of media. 12 h after LPS challenge, cells were harvested, and total protein was extracted and used for multianalyte ELISA. LPS-treated TDP-43WT transgenic microglia had significantly higher levels of TNF (2.5-fold; P < 0.01), IL-1β (2.3-fold; P < 0.01), IL-6 (twofold; P < 0.05), and IFN-γ (twofold; P < 0.05) as compared with LPS-treated microglia from C57BL/6 nontransgenic mice (). However, in the absence of LPS stimulation, no significant differences in cytokines levels were detected between microglia from TDP-43WT transgenic mice and from nontransgenic mice (not depicted). The p65 level was significantly higher (threefold; P < 0.01) in LPS-treated TDP-43WT microglia as compared with nontransgenic microglia (). We also treated primary microglial cultures with 1 mM H2O2 for 1 h (and incubated in serum-free media for 12 h) to study the effect of reactive oxygen species (ROS) on primary microglial cultures. H2O2-treated TDP-43WT transgenic microglia had significantly higher levels of TNF (threefold; P < 0.01), IL-1β (2.5-fold; P < 0.01), IL-6 (1.7-fold; P < 0.05), IFN-γ (twofold; P < 0.05), and p65 (RELA) levels (2.2-fold; P < 0.05) when compared with H2O2-treated microglia from C57BL/6 nontransgenic mice () as determined by multianalyte ELISA.
LPS stimulation of primary microglial cells caused degradation of IκB-α as shown in . The decrease in IκB-α levels was more pronounced in microglia overexpressing TDP-43 species. After LPS treatment, the increases in levels of p65, phospho-p65Ser536, p50, and phospho-p50Ser337 were also more robust in transgenic microglia overexpressing TDP-43 species (). Similarly, H2O2 treatment led to a reduction in IκB-α levels and increase in levels of p65 and phospho-p65Ser536 in TDP-43WT (). Again, the effects were more pronounced in transgenic microglia overexpressing TDP-43 species (). We then treated primary astrocytes with LPS and studied their response to LPS using real-time RT-PCR. LPS-treated TDP-43WT transgenic astrocytes had significantly higher levels of IL-α (1.75-fold; P < 0.05), IL-1β (1.67-fold; P < 0.05), IL-6 (2.8-fold; P < 0.01), IL-18 (1.8-fold; P < 0.05), and chemokines like CSF (1.6-fold; P < 0.05), CCL5 (1.9-fold; P < 0.05), and CXCL12 (2.67-fold; P < 0.01) as compared with LPS-treated microglia from C57BL/6 nontransgenic mice ().
To further evaluate the innate immune response in TDP-43WT transgenic mice, we isolated BMMs from TDP-43WT transgenic mice and from C57BL/6 nontransgenic mice. In LPS-stimulated TDP-43WT macrophages, there was an increase of 1.6-fold (P < 0.05) in TLR2 mRNA levels, 1.8-fold (P < 0.05) in MyD88 levels, and 2.6-fold (P < 0.01) in p65 (RELA; P < 0.01) levels as compared with LPS-stimulated control (nontransgenic) macrophages (). We also found in LPS-stimulated TDP-43WT macrophages that there was an increase of 3.2-fold (P < 0.01) in TNF, 3.5-fold in IL-1β (P < 0.01), and 2.6-fold in IL-12p40 levels, 2.5-fold (P < 0.01) in IL-6 levels, twofold (P < 0.05) in Cox-2 and iNOS levels, threefold in IP-10 levels (P < 0.01), and 2.1-fold in RANTES (P < 0.05) mRNA levels as compared with LPS-stimulated control (nontransgenic) macrophages ().
TDP-43 up-regulation increases microglia-mediated neurotoxicity
We then examined the effect of TDP-43 overexpression on toxicity of microglia toward neuronal cells. This was done with the use of primary microglia and of cortical neurons derived from transgenic mice overexpressing TDP-43 species (TDP-43WT, TDP-43A315T, or TDP-43G348C) and C57BL/6 nontransgenic mice. Primary cortical neurons were cultured for 12 h in conditioned media from LPS-stimulated microglial cells. All conditioned media from LPS-challenged microglia increased the death of cortical neurons in culture (). The media from LPS-stimulated nontransgenic microglial cells increased the neuronal death of nontransgenic mice by 3.5-fold (P < 0.01). However, there were marked increases of neuronal death caused by conditioned media from LPS-challenged microglia (of same genotype) overexpressing TDP-43 species: 5.5-fold (P < 0.001) for TDP-43WT, 6.5-fold (P < 0.001) for TDP-43A315T, and 7.5-fold (P < 0.001) for TDP-43G348C. The increased neurotoxicity of the conditioned media was associated with increased ROS and NO production. The ROS production, as determined by H2DCFDA fluorescence, was significantly higher in conditioned media–challenged neurons from TDP-43WT (1.5-fold; P < 0.05), TDP-43A315T (1.8-fold; P < 0.05), or TDP-43G348C (twofold; P < 0.05) as compared individually with conditioned media–challenged nontransgenic control neurons (). Similarly, the nitrite (NO) production was significantly higher in TDP-43WT (1.5-fold; P < 0.05), TDP-43A315T (2.3-fold; P < 0.05), or TDP-43G348C (threefold; P < 0.01) as compared individually with nontransgenic control ().
Inhibition of NF-κB activation reduces vulnerability of TDP-43–overexpressing neurons to toxic injury
The aforementioned experiments also revealed that the presence of TDP-43 transgenes in cortical neurons increased their vulnerability to microglia-mediated toxicity. NF-κB is known to modulate p53-p38MAPK–dependent apoptosis in neurons when treated with DNA damage–inducing chemicals like camptothecin (Aleyasin et al., 2004
), glutamate excitotoxicity (Pizzi et al., 2005
), or general bystander-mediated killing of neurons by microglia (Sephton et al., 2010
). To assess the potential contribution of NF-κB to the death of TDP-43–overexpressing neurons exposed to toxic injury, we prepared cultures of primary cortical neurons and microglia from transgenic mice overexpressing TDP-43WT
or TDP-43 mutants. Cortical neurons were exposed to 10 µM glutamate for 15 min, with or without 1 µM Withaferin A (WA), a known inhibitor of NF-κB (Oh et al., 2008
). The lactate dehydrogenase (LDH) cytotoxicity was determined 24 h later (see ). We found that neurons overexpressing TDP-43 species were more vulnerable than nontransgenic neurons to glutamate cytotoxicity and that inhibition of NF-κB by WA resulted in a marked decrease in cell death: TDP-43WT
(twofold; P < 0.01), TDP-43A315T
(threefold; P < 0.01), and TDP-43G348C
(threefold; P < 0.01). The addition of WA inhibited NF-κB, as detected by reduced levels of phospho-p65Ser536
(see ). We then incubated cortical neurons with the conditioned media from primary microglial culture, which were challenged with LPS at a concentration of 100 ng/ml of media. Treatment of neuronal cultures with WA resulted in substantial decrease in microglia-mediated death of neurons overexpressing TDP-43WT
(twofold; P < 0.01), TDP-43A315T
(threefold; P < 0.01), or TDP-43G348C
(threefold; P < 0.01). As WA might exert multiple pharmacological actions, we tested a more specific molecular approach for inhibiting NF-κB. Because activation of NF-κB requires its dissociation from the inhibitory molecule, IκB, we expressed a stable mutant super-repressive form of IκB-α (Ser32/Ser36 to alanine mutant; IκBSR
) and evaluated its effects on neuronal death. Cultured cortical neurons from TDP-43 transgenic and nontransgenic mice were transfected with a plasmid construct, expressing IκBSR
, and exposed to either 10 µM glutamate for 30 min or incubated in conditioned media from LPS-stimulated microglia of the same genotype. Similar to WA treatment, we found that IκBSR
inhibited NF-κB activation and it attenuated the glutamate-induced or microglia-mediated death of neurons overexpressing TDP-43WT
(1.3-fold; P < 0.01), TDP-43A315T
(1.5-fold; P < 0.01), and TDP-43G348C
(twofold; P < 0.01; ).
Figure 10. WA ameliorates TDP-43–mediated toxicity. (A) Primary cortical neurons from TDP-43WT, TDP-43A315T, TDP-43G348C, and B6 nontransgenic mice were exposed to 10 µM glutamate for 15 min or incubated in conditioned media from LPS-stimulated microglia (more ...)
Figure 9. Inhibition of NF-κB reduces neuronal vulnerability to toxic injury and ameliorates disease phenotypes in TDP-43 transgenic mice. (A) A stable mutant super-repressive form of IκB-α (IκBSR) was expressed, and its effects (more ...)
NF-κB inhibition by WA treatment reduces inflammation and ameliorates motor impairment of TDP-43 transgenic mice
To study the in vivo effect of NF-κB inhibition on disease progression, we injected TDP-43WT
;GFAP-luc double transgenic mice with 3 mg/kg body weight of WA twice a week for 10 wk starting at 30 wk. The pharmacokinetic parameters of WA have been published recently (Thaiparambil et al., 2011
), and we have determined that this compound passes the blood–brain barrier (unpublished data). We used TDP-43WT
;GFAP-luc double transgenic mice because the reporter luciferase allowed the longitudinal and noninvasive biophotonic imaging with charge-coupled device camera of the GFAP promoter activity, which is a target of activated NF-κB. To analyze the spatial and temporal dynamics of astrocyte activation/GFAP induction in the TDP-43 mouse model, we performed a series of live imaging experiments. These live imaging experiments revealed that treatment of TDP-43WT
;GFAP-luc mice with WA caused progressive reduction in GFAP-luc expression in the spinal () compared with untreated TDP-43WT
mice, which continued to exhibit high GFAP-luc expression. The down-regulation of GFAP promoter activity was further confirmed in these mice using GFAP immunofluorescence of spinal cord sections of TDP-43WT
mice (both drug treated and untreated; ).This down-regulation of GFAP in WA-treated mice was actually caused by a reduced amount of active p65 in the nucleus of cells as indicated by p65 EMSA (). Down-regulation of GFAP along with reduction in active p65 levels in WA-treated mice prompted us to analyze behavioral changes in these mice. Analysis of motor behavior using accelerating rotarod showed that WA-treated TDP-43WT
mice had significantly better motor performance compared with untreated TDP-43WT
mice as indicated by improved rotarod testing times (). We performed peripherin immunofluorescence and found reduction of peripherin aggregates in WA-treated TDP-43WT
mice (). Peripherin levels were also reduced in WA-treated TDP-43WT
mice as seen by immunoblot (). Double immunofluorescence of activated microglial marker Mac-2 and Cox-2 showed a marked reduction in activated microglia in WA-treated TDP-43WT
mice ( and ). The WA-treated mice also had a 40% reduction in the number of partially denervated neuromuscular junctions (NMJs; ).