Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-β-induced Neuronal Death*

Amyloid-β (Aβ)-induced neuron death is considered central to the pathogenesis of Alzheimer's disease (AD). Among several death modalities, autophagy and apoptosis play important roles in Aβ-induced neuron death suggesting that there may be regulatory mechanisms that initiate both cell death pathways. However, molecules that govern both pathways have not been identified. Here, we report that, upon Aβ treatment, tribbles pseudokinase 3 (Trib3, an ortholog of Drosophila Tribbles) is up-regulated in neurons both in vivo and in vitro. Increased Trib3 levels inhibited the activity of the kinase Akt by interacting with it. As a result, forkhead box O1 (FoxO1), a transcription factor that is negatively regulated by Akt, was activated, translocated to the nucleus, and induced the pro-apoptotic gene BCL2-like 11 (Bim). Conversely, FoxO1 responded to Aβ insult by binding to the Trib3 gene promoter, enhancing its expression. Our investigations further revealed that Trib3 also induces autophagy. We found that Trib3 indirectly activates unc-51-like autophagy-activating kinase1 (Ulk1) by impeding phosphorylation of, and thus inactivating, a negative regulator of Ulk1, mechanistic target of rapamycin. Ulk1 activation augmented autophagosome formation and reduced autophagy flux. Thus, Trib3 was required for formation of autophagosomes, which accumulated in neurons as autophagic flux was thwarted. Most importantly, silencing endogenous Trib3 strongly protected neurons from Aβ insult. Our results suggest that a self-amplifying feed-forward loop among Trib3, Akt, and FoxO1 in Aβ-treated neurons induces both apoptosis and autophagy, culminating in neuron death. Thus, Trib3 may serve as a potential therapeutic target for AD.

Alzheimer's disease (AD) 2 is a progressive neurodegenerative disorder that is emerging as the leading cause of dementia today. It involves a gradual deterioration in several cognitive domains, including loss of memory. The two pathognomonic features of the disease include plaques of ␤-amyloid (A␤) and neurofibrillary tangles of hyperphosphorylated Tau (1,2). The disease is also characterized by accumulation of misfolded proteins, compromised autophagy, enhanced oxidative stress, and metabolic perturbations (3)(4)(5). Disturbances in cellular homeostasis lead to increased endoplasmic reticulum (ER) sensitivity, which is a characteristic feature in AD. Excessive ER stress is detrimental to neurons because it can switch on an apoptotic program and also can trigger inflammatory responses (6). In contrast, inappropriate aggregation of waste proteins and shoddy organelles induce autophagy via ER stress (7). Mounting evidence reveals the presence of accumulated autophagosomes and defective autophagy flux in the pathogenesis of AD (8,9). It has also been demonstrated that A␤ accumulates in autophagic vacuoles, and inappropriate clearance of these vacuoles, impaired autophagic-lysosomal degradation, promotes the extracellular deposition of it thus exacerbating the pathological condition in AD (10 -13).
Recent studies indicate the dynamic participation of ER stress in activating autophagy and promoting apoptosis of tumor cells, wherein Trib3 (Tribbles homolog 3) leads the saga by inhibiting Akt and mTORC1 in turn leading to enhanced autophagy and subsequent apoptosis in hepatocarcinoma cells and glioma cells (14,15). Trib3 is a mammalian ortholog of the Drosophila Tribbles gene and is also known as neuronal deathinducible putative kinase/Sink1/Skip3 (16). Trib3 is responsible for a plethora of functions ranging from glucose regulation, migration of tumor cells, suppressing differentiation of adipocytes, and cell cycle control (17)(18)(19)(20). It was identified as a novel ER stress-inducible gene that, when up-regulated, activated several genes involved in cell death during ER stress (21). Trib3 is also shown to be elevated by several stresses, including hypoxia, 6-hydroxydopamine, growth factor deprivation, anoxia, and ethanol exposure (16,(22)(23)(24)(25)(26)(27)(28). It has also been shown that Trib3 is elevated in Parkinson's disease brains and mediates neuron death in various Parkinson's disease models (27). Trib3 is a pseudokinase because it lacks the catalytic residues required for its kinase function (29,30). Bioinformatic analysis of Trib3 protein reveals the presence of a number of conserved domains that account for its ability to interact with numerous protein-binding partners (25,(31)(32)(33).
AD has well been characterized as a multifactorial disease where a single unwavering approach to tackle the disease might be ineffective. A combination of treatment strategies may prove beneficial in this arena. Several approaches have been studied, yet most of them have met with failure at the stage of clinical trials. Because the A␤ cascade hypothesis holds the spotlight of the pathogenesis of the disease, targeting A␤ proves to be a promising approach (34,35). Apart from this, a complementary therapy is imperative to impede the toxicity due to A␤, the complete removal of which is difficult. Hence, a complete understanding of the molecular mechanism of A␤-induced death is quintessential. In this study, we have investigated the role of Trib3 in neuronal death induced by A␤. It appears that Trib3 is induced and promotes death of neurons by both apoptosis and autophagy in response to A␤.

A␤ Treatment Induces Trib3 mRNA and Protein Levels in
Vitro and in Vivo-Accumulating evidence implicates A␤ oligomers as the principal cause of AD pathogenesis (36,37). Oligomeric A␤ at a concentration of 1.5 M leads to significant death of primary cortical and hippocampal neurons after 24 h of exposure (38). We determined the levels of Trib3 in neurons after A␤ exposure. We found that Trib3 levels were increased in cultured cortical neurons following A␤  treatment. To check the specificity of the action of A␤(1-42), we used a reverse peptide, A␤ , and we found that the reverse peptide A␤(42-1) has no effect on Trib3 levels in the primary cortical neurons (data not shown). Trib3 transcript levels were significantly increased as early as after 4 h and about 3-fold increased after 8 h of A␤(1-42) treatment as detected by semiquantitative (Fig. 1A) and real time PCR (Fig. 1B). Protein levels of Trib3 were also significantly increased within 4 h and they were about 3-and 3.5-fold increased after 8 and 16 h of A␤ treatment, respectively ( Fig. 1, C and D). Thus, Trib3 expression was elevated well before cell death became apparent (see below and Fig. 2C).
Next, we investigated whether this increase of Trib3 in vitro is also reflected in in vivo conditions. Reports reveal that oligomeric A␤ when infused into adult rat brains results in A␤ deposition, caspase-3 activation, and neuronal cell loss in the vicinity of A␤ infusion (38). In our study adult rats were infused with A␤ or PBS on the right hemisphere of their brains. 21 days later these animals were sacrificed, and their brains were fixed, cryosectioned, and then immunostained with A␤(1-42) antibody to check A␤ deposition in the site of A␤ infusion (Fig. 1E). Adjacent sections were also co-immunostained with Trib3 and NeuN (a neuronal marker) antibodies. Nuclei were stained with Hoechst dye. Results revealed marked up-regulation of Trib3 in A␤-infused rat brains as compared with PBS-infused rat brains (Fig. 1, F and G). Because synthetic A␤ may behave differently from naturally secreted A␤, brain sections of APPswe-PS1de9 (Swedish mutation in amyloid precursor protein and PS1 mutation) transgenic mice, which naturally secrete A␤, were also examined for Trib3 expression. The presence of A␤ plaques in the transgenic mouse brain was checked by Congo red staining (Fig. 1H). Transgenic and control littermate mouse brains were cryosectioned and co-immunostained with Trib3 and NeuN antibodies. Hoechst dye was used to stain the nuclei. It was observed that there was a significant increase in Trib3 levels in transgenic mice as compared with the control littermates ( Fig.  1, I and J). Therefore, our study indicates that Trib3 expression is increased in neurons upon exposure to A␤ in vitro and in vivo. To find the role of Trib3 in A␤-induced neuronal cell death in vivo, we performed immunostaining of Trib3 along with TUNEL assay in sections in the vicinity of A␤ infusion. We observed that increased Trib3 expression co-localized with TUNEL-positive cells in A␤-infused rat brain sections as compared with PBS-infused rats (Fig. 1K, see arrows). This indicates the role of Trib3 in A␤-induced neuronal cell death in vivo.
Trib3 Plays an Essential Role in Neuronal Death Evoked by A␤-We next assessed whether Trib3 is necessary in evoking neuronal death in response to A␤. We interfered with the expression of Trib3 using previously described shRNA constructs (23). The Trib3 shRNA construct efficiently blocked induction of Trib3 by A␤ in neuronally differentiated (primed) PC12 cells (Fig. 2, A and B). Primary cultured cortical neurons were then transfected with this shRNA construct (shTrib3) or a control shRNA construct (shRand) and maintained for 48 h followed by oligomeric A␤ treatment (1.5 M). Transfected live green cells were monitored and counted under a fluorescence microscope at different time intervals. Down-regulation of Trib3 by shRNA blocked neurodegeneration as evident from the retention of neuronal processes and caused significant survival of these neurons compared with shRand-transfected neurons even after 72 h of A␤ treatment (Fig. 2, C and D).
A similar experiment with cultured hippocampal neurons revealed that down-regulation of Trib3 also provided significant protection of hippocampal neurons from A␤-induced neurotoxicity (Fig. 2, E and F). Hippocampal neurons expressing shTrib3 not only showed increased viability as compared with shRand-transfected neurons but also displayed enhanced retention of neurites and overall neuronal morphology (Fig. 2, E and F). Furthermore, we quantitatively assessed the retention of neurites and neuronal networks in shTrib3-transfected hippocampal neurons after A␤ treatment by Sholl analysis as described (38,39). Single hippocampal neurons transfected with either shTrib3 or shRand were analyzed by ImageJ (National Institutes of Health) as described under "Experimental Procedures." Results revealed that the number of crossings remained almost same before and after treatment of A␤ in the case of shTrib3-transfected neurons, although there was a drastic reduction in the number of crossings in shRand-transfected neurons (Fig. 2G). Furthermore, we found that down-regulating Trib3 retains mitochondrial membrane potential (Fig. 2H), maintains post-synaptic membrane integrity (Fig. 2I), and decreases nuclear localization of cleaved caspase3 (Fig. 2J) even in A␤-treated conditions. We also found a similar protective effect of shTrib3 on neuronal PC12 cells upon A␤ treatment (data not shown). Taken together, these results suggest that Trib3 plays a necessary role in mediating neuron degeneration and death evoked by A␤ toxicity.
Trib3 Negatively Regulates Akt upon A␤ Treatment-It has been shown that Trib3 blocks Akt activity by physically interacting with it in various non-neuronal cells (40 -42). As phospho-Akt level is reported to be markedly reduced in AD brain (43), we tested whether Trib3 also negatively regulates Akt in neuronal cells in response to A␤. Primary cultures of cortical neurons were treated with A␤, and cell lysates were immunoprecipitated with Trib3 antibody and Western blotted with Akt antibody and vice versa. Results revealed increased binding of Trib3 with Akt in A␤-treated neurons compared with control neurons (Fig. 3, A and B). We then checked the phosphorylation status of Akt upon A␤ treatment. We observed that the decrease in phosphorylation at Ser-473 of Akt caused by A␤ insult was rescued when Trib3 was down-regulated (Fig. 3, C and D). We also observed that when PI3K is inhibited by a specific PI3K inhibitor in the presence or absence of A␤, the protein levels of Trib3 increased even in the absence of A␤ (Fig.  3, E and F). This allowed us to see whether Trib3 is regulated by some downstream substrate of PI3K/Akt, which gets activated upon PI3K inhibition and in turn regulates Trib3. Transcription factor FoxO, downstream to PI3K/AKT and an entrenched substrate of it, is known to translocate from the cytosol to FIGURE 1. Trib3 mRNA and protein levels are elevated in response to A␤ in vitro and in vivo. Primary rat cortical neurons (7DIV) were treated with oligomeric A␤ 1.5 M for the indicated times. A, total RNA was isolated, subjected to reverse transcription, and analyzed by semi-quantitative PCR using Trib3 primers. GAPDH was used as loading control. B, graphical representation of fold changes in Trib3 transcript level upon A␤ treatment to rat cortical neurons for the indicated times by quantitative real time PCR. GAPDH was used as loading control. Data represent mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05; **, p Ͻ 0.01. C, primary cultured rat cortical neurons were treated with A␤ for the times indicated. Total cell lysates were subjected to Western blotting analysis for Trib3 levels. A representative immunoblot of three independent experiments with similar results is shown. Actin was used as loading control. D, graphical representation of the Trib3 protein levels as quantified by densitometry of Western blottings in cortical neurons subjected to A␤ treatment for different time points. Data are expressed relative to untreated control. Data represents mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05; **, p Ͻ 0.01. E, brain sections of the infused rats were immunostained with A␤(1-42) antibody to check the presence of A␤ plaques in the infused brain area. Upper panel shows immunostaining of A␤(1-42) antibody from brain sections of rats infused with PBS. Lower panel shows brain section immunostained with A␤(1-42) antibody of A␤-infused rat brains. F, adult rat brains were infused with either A␤ or PBS in the right hemispheres. 21 days later, the animals were sacrificed; brains were then cryosectioned, and sections were co-immunostained with antibodies for Trib3 and NeuN. Hoechst dye was used to stain nuclei. A representative image of one of the brain sections with similar results in each case is shown here. Images were taken using an inverted fluorescence microscope. G, graphical representation of corrected total cell fluorescence of Trib3 in PBS-infused and A␤-infused brain sections. Difference in the intensity of Trib3 staining is quantified by ImageJ as described under "Experimental Procedures." Data represent mean Ϯ S.E. of 30 different cells from three independent experiments. **, p Ͻ 0.001. H, Congo red staining of brain slices from transgenic and wild-type mice. Transgenic mouse brain shows the presence of amyloid plaques (shown by arrow), whereas such plaques were absent in the wild-type brain tissue. Representative images of six sections from three animals of each group with similar results are shown here. Images were taken under ϫ5 objective. I, level of Trib3 expression was analyzed in brain sections obtained from A␤PPswe-PS1de9 transgenic mice and control littermates. Brain sections from transgenic and control mice were co-immunostained with Trib3 and NeuN antibodies. Nuclei were stained with Hoechst. Representative image of one of the brain sections with similar results in each case is shown here. J, graphical representation of corrected total cell fluorescence of Trib3 in transgenic and wild-type brain sections. Difference in intensity of Trib3 staining is quantified by ImageJ as described under "Experimental Procedures." Data represent mean Ϯ S.E. of 30 different cells from three independent experiments. **, p Ͻ 0.001. K, brain sections from A␤ and PBS infused rats were analyzed for TUNEL assay and then immunostained for Trib3. Nuclei were stained with Hoechst. Representative images of one of the brain sections with similar results in each case is shown. nucleus and induces its target genes in A␤-treated neurons (38,44). Previous reports reveal the presence of putative FoxObinding sites on the Trib3 promoter (45)(46)(47)(48). We therefore hypothesized that Trib3 may be regulated by FoxO transcription factors in A␤-treated neurons. We performed chromatin immunoprecipitation assay to check the direct binding of FoxO1 with the Trib3 promoter. The result showed that FoxO1 occupancy of the Trib3 promoter was markedly increased in response to A␤ (Fig. 3, G and H). These results indicate that Trib3 directly binds and inactivates Akt; this in turn activates the transcription factor FoxO1, and FoxO1 can occupy the Trib3 gene promoter upon A␤ treatment.
Feed-forward Regulatory Mechanism Acts between Trib3 and FoxO1-Previous results prompted us to test whether a feedforward regulatory mechanism acts between Trib3 and FoxO in neuronal cells in response to A␤ insult. To study the regulation of Trib3 by FoxO, we knocked down the FoxOs in cortical neurons by a previously reported shRNA that targets all FoxO isoforms (49) and exposed them to A␤ and performed immunocytochemistry to see the endogenous Trib3 expression. Results revealed that the shRNA-mediated knockdown of FoxOs led to reduced expression of Trib3 in most of the transfected cells compared with non-transfected neighboring cells upon exposure of A␤ (Fig. 4A). In contrast, no such reduction on Trib3 expression was found in shRand-transfected cells (Fig.  4A). A quantitative analysis showed that about 80% shFoxO and about 20% shRand-transfected neurons had reduced Trib3 levels upon A␤ treatment (Fig. 4B). To confirm this result, we performed a Western blotting analysis of A␤-treated shFoxO and shRand-transfected cells, and we observed the Trib3 protein levels. We observed that downregulating FoxOs blocked induction of Trib3 protein caused Representative pictures of transfected neurons that were maintained in the presence or absence of A␤ for the indicated time periods are shown. Images were taken using an inverted fluorescence microscope. D and F, graphical representation of percentage of viable green cells after each time point. Numbers of surviving transfected (green) cells were counted under fluorescence microscope just before A␤ treatment and after 24, 48, and 72 h of the same treatment. Data are from three independent experiments, each with comparable results, and are shown as mean Ϯ S.E., performed in triplicate. The asterisks denote statistically significant differences from control (shRand) at corresponding time points: *, p Ͻ 0.05; **, p Ͻ 0.001. G, Trib3 knockdown prevents neuronal degeneration and preserves neuritic processes. Sholl analysis of single imaged neurons by using ImageJ was done as described under "Experimental Procedures." Data represent mean Ϯ S.E. of six different neurons from three independent cultures for each class. Asterisks denote statistically significant differences from shRand (control): *, p Ͻ 0.001. H-J, cultured cortical neurons (5DIV) were transfected with shTrib3 and shRand, and the cells were maintained for the next 48 h and then treated with 1.5 M A␤ for 16 h, after which they were immunostained with MitoTracker red dye (H), PSD95 (I), or cleaved caspase3 antibodies (J).
by A␤ toxicity (Fig. 4, C and D). This indicates that FoxOs act as regulator of Trib3.
Next, we investigated whether Trib3 regulates FoxO1 in our model. Cortical neurons were transfected with shTrib3, treated with A␤ and immunostained to study FoxO1 expression. It has already been reported that nuclear translocation of FoxO1 is controlled by Trib3 in anti-NGF-treated neurons (23). In our study we found that shTrib3 not only blocked translocation of FoxO1 but also reduced its protein levels (Fig. 4, E and F). To confirm our result, we transfected neuronal PC12 cells with shTrib3 and shRand, treated with A␤ for 16 h, and performed Western blotting analysis for FoxO1 protein. Results confirmed that down-regulation of Trib3 led to reduced levels of FoxO1 in the presence or absence of A␤ (Fig. 4, G and H). Thus, our findings indicate that Trib3 regulates both the level and activity of FoxO1 following A␤ treatment. Collectively, these results suggest a feed-forward loop that involves inhibition of Akt by Trib3, activation of transcription factor FoxO1 upon inactivation of Akt, and transactivation of Trib3 by FoxO1 in A␤-treated neurons.
Trib3 Further Downstream Regulates Pro-apoptotic Gene Bim in Neuronal Cell Death Induced by A␤-It has also been reported that transcription factor FoxO regulates Bim upon NGF deprivation in sympathetic neurons (50). We have recently shown that Bim is directly regulated by FoxO in neurons following A␤ treatment (38). We therefore determined whether Bim is under the control of Trib3 in this death paradigm. We transfected cultured cortical neurons with shTrib3 or FIGURE 3. Trib3 binds to Akt and negatively regulates it. A, cultured rat cortical neurons were treated with 1.5 M A␤ for 16 h; cell lysate was immunoprecipitated (IP) with Trib3 antibody and immunoblotted (IB) with Akt antibody; reverse immunoprecipitation was also performed to check interaction of Trib3 with Akt; cell lysate was also immunoprecipitated with p-Akt and immunoblotted with Trib3 (2nd column). Immunoprecipitation with nonspecific antibody was done to check specificity of the reactions (3rd column). Actin was used as loading control (4th column). Levels of the specific proteins in cell lysates (Inputs) were also checked (5th column). Immunoblot of the respective immunoprecipitated protein was also performed (1st column). Lane C, control. B, graphical representation of the Akt protein levels as quantified by densitometry of Western blottings. Data represents mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05. C, primed PC12 cells were transfected with shTrib3 or shRand, treated with and without A␤ 5 M. Levels of endogenous p-Akt(Ser-473) were assessed by Western blotting analysis using anti-p-Akt(Ser-473) antibody. D, graphical representation of fold change of p-Akt(Ser-473) levels by densitometric analysis of Western blottings. Data represent mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05; **, p Ͻ 0.005. E, cultured cortical neurons were treated with A␤ 1.5 M for 16 h, with and without LY 294002 (PI3Ki). Total cell lysates were subjected to Western analysis with Trib3 antibody. A representative immunoblot of three independent experiments with similar results is shown. F, graphical representation of fold change of Trib3 upon A␤ treatment with and without PI3Ki. Data represent mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05. G, primary cultures of rat cortical neurons were treated with or without A␤ for 8 h. An equal number of cells were processed for ChIP assay using anti-FoxO1 antibody for immunoprecipitation. The immunoprecipitated materials were subjected to PCR using primers against the region of Trib3 promoter that contains the putative FoxO1-binding site. PCR products were verified by agarose gel electrophoresis. Templates were DNA from cells before ChIP (input) or DNA from immunoprecipitated (IP) materials. PCR assays were conducted after ChIP using samples from cells that were either left untreated (control) or treated with A␤. ChIP assay indicates that FoxO1 occupancy of rat Trib3 promoter regions enhances after A␤ exposure. H, graphical representation of fold changes in FoxO1 association with the Trib3 promoter upon A␤ treatment. Data represent mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05. shRand and exposed them to A␤ followed by immunocytochemical analysis for Bim expression. Results revealed that down-regulating Trib3 resulted in a marked reduction of Bim expression, as evident from reduced Bim staining in shTrib3transfected cells as compared with shRand-transfected cells (Fig. 5, A and B). To further corroborate our findings, we performed Western blotting analysis with Trib3 knockdown PC12 cells for Bim levels following A␤ treatment. We observed a significant up-regulation of Bim following A␤ exposure in shRand-transfected cells as expected. But interestingly, this upregulation was significantly blocked in shTrib3-transfected cells even after A␤ treatment (Fig. 5, C and D). These findings thus indicate that Trib3 induces neuronal death upon A␤ exposure by activating Bim, which could be via the Akt-FoxO1 pathway.

Trib3 Induces Autophagy in Neurons via the Akt-mTOR
Pathway-Recently, it has been reported that Trib3 induces autophagy in cancer cells in response to cannabinoid by inhibiting the Akt/mTORC1 axis (14,15). Multiple studies implicate impaired autophagy in the pathogenesis of AD (9 -13). A␤ treatment can induce both apoptosis and autophagy in neuronal cells (data not shown) (10,12,35,51). Therefore, we investigated whether Trib3 also induces autophagy in A␤-treated neuronal cells. We transfected differentiated PC12 cells with shTrib3 or shRand and followed it with A␤ treatment. Western blotting analysis was performed to check the level of phospho-mTOR at Ser-2448 (p-mTOR) and total mTOR. We observed that upon treatment, p-mTOR levels decrease, and this decrease in phosphorylation level can be blocked by down-regulating Trib3 (Fig. 6, A and B). A direct target of p-mTOR is Ulk1 (52). mTOR when phosphorylated leads to the inactivating phosphorylation of Ulk1 at Ser-757. This results in reduced autophagy. We therefore analyzed the phosphorylation status of the Ulk1 protein. We found that Ulk1 phosphorylation at Ser-757 was reduced upon A␤ treatment and rescued by down-regulating Trib3 (Fig. 6, C and D). Analysis of pUlk1 levels in transgenic mice revealed that there is decreased expression of pUlk1(Ser-757) in the transgenic mouse brain as compared with the brain of wild type (Fig. 6, E and F). Taken together, these results indicate that Trib3 when induced upon A␤ treatment leads to enhanced levels of autophagy by inhibiting mTOR and activating Ulk1 activity.
Trib3 Induces Frustrated Autophagy in Neurons-Although a basal level of autophagy is required for protein degradation and organelle turn over, reports indicate that increased autophagy facilitates enhanced clearance of aggregated proteins, thus promoting survival of neurons (11,12). However, if there is an incessant up-regulation of autophagic activity, it can lead to cell death (53). Autophagic death is said to occur not just due to increased autophagosome formation but also when there is a dysregulation of autophagic flux. Such an autophagy is referred to as frustrated autophagy (54). To check the efficiency of autophagy induced by Trib3, we monitored the levels of LC3 after A␤ exposure. We found that upon A␤ exposure there was an increase in LC3 punctate staining in transgenic mouse brain sections as compared with wild-type mice (Fig. 6, G and H). The levels of both LC3-I and LC3-II in the cells have also increased in response to A␤ treatment (Fig. 6, I and J). However, there was a significant reduction in the levels of LC3-II when Trib3 was down-regulated (Fig. 6, I-L). It has been reported that p62 is accumulated when autophagic degradation is inhibited and is therefore considered a marker of autophagy flux (55,56). We checked the autophagy flux in A␤-treated cells by monitoring p62 levels. Interestingly, we found that there was an increased accumulation of p62 in the transgenic mouse brain as compared with the wild-type mouse brain (Fig. 7, A and B). The level of p62 was also increased in cells treated with A␤ (data not shown). Conversely, the increase in p62 levels in these cells was blocked when Trib3 was down-regulated (Fig. 7, C-F). In contrast, when Trib3 was overexpressed, an enhanced accumulation of p62 was observed in the cells (Fig. 7, G and H). Collectively, these results indicate that Trib3 induces autophagy in A␤-treated cells. However, it culminates in a frustrated autophagy due to decreased autophagic flux as seen by accumulation of p62.
A␤-induced Impaired Autophagy Flux Contributes to Neuron Death-Our results suggest that Trib3 up-regulation upon A␤ exposure leads to induction of both apoptosis and autophagy. We determined the contribution of these two processes on neuron death evoked by A␤. We found that inhibition of both these processes separately blocks the death of neuronal PC-12 cells substantially. Moreover, inhibition of both apoptosis and autophagy provided better protection to these cells from A␤ toxicity (Fig. 8, A and B). Thus, our results suggest that simultaneous induction of apoptosis and autophagy by A␤ leads to neuronal cell death.

Discussion
In this study, we investigated the role of Trib3 in A␤-induced neurodegeneration. A number of our experimental observations indicate that Trib3 promotes neuronal death by both apoptosis and autophagy in response to A␤. We found that Trib3 expression is up-regulated in neuronal cells both at transcriptional and translational levels following A␤ treatment. A similar induction of Trib3 was also found in the A␤-infused rat model. Because we used synthetic A␤ for our experiments, which could have disparate effects from the naturally occurring A␤, we also examined the induction of Trib3 in the transgenic mouse brain that overexpresses human A␤. The result from these transgenic mice also showed an elevated level of Trib3 protein corroborating our previous in vitro results. Moreover, knocking down Trib3 by using shRNA in cortical or hippocampal neurons pro-tects cells from A␤-induced cell death. Sholl analysis further exhibits the retention of neuronal processes as well as preservation of the overall neuronal morphology in Trib3 knocked down neurons, even after A␤ treatment.
An important question is how A␤ applied in media affects different organelles in the cells. Reports suggest that A␤ from the medium is internalized by the PC12 cells, enters their nuclei (57), and leads to death of PC12 cells (58). Studies also reveal that A␤ enters neurons and is present in their lysosomal system (59), whereas inhibition of its engulfment reduces death of neurons (60). A␤ immunoreactivity has also been found in degenerating neurons in transgenic mice and AD brains (61,62).
Mechanistic studies reveal that Trib3 interacts and negatively regulates survival kinase Akt. These results corroborate previous results whereby overexpression of Trib3 reduced phos- phorylation of Akt at Ser-473 and Thr-308 (23) and knockdown of Trib3 restored phosphorylation of Akt in tunicamycintreated PC12 cells (28). Interestingly, it has recently been shown that Trib3 induction, Akt inhibition, and FoxO activation are linked in a self-amplifying loop that culminates in neuron death evoked by NGF deprivation (23). We have also found that inhibition of PI3K/Akt signaling by LY294002 led to upregulation of Trib3. In recent years, FoxO transcription factors have been shown to play an important role in neurodegeneration (38,44). Reports suggest putative FoxO-binding sites on the Trib3 promoter (45)(46)(47)(48). Our results revealed an enhanced occupancy of FoxO1 on the Trib3 gene that regulated its expression upon A␤ insult. These findings clearly indicate that a feed-forward regulatory mechanism is active between Trib3, Akt, and FoxO1 in A␤-treated neuronal cells as well. Next, we investigated the downstream target of up-regulated Trib3 that mediates neuronal death. We observed that down-regulating Trib3 abrogates the up-regulation of Bim upon A␤ exposure suggesting the potential role of Bim in the mechanism by which Trib3 synchronizes apoptotic death of neurons. Bim is a FoxO target and has also been implicated in neuronal apoptosis in AD (49,(63)(64)(65).
Interestingly, we found that neuronal death caused by Trib3 overexpression could not be fully rescued by inhibiting caspases. 3 This aroused our curiosity to investigate the possibility of the existence of a varied mechanism by which Trib3 could be triggering cell death. Reports revealed that Trib3 was responsible for autophagic death in cancer cells (6,7,14). Autophagic failure in AD has also been studied extensively 3 S. Saleem and S. C. Biswas, unpublished observations. FIGURE 7. A␤ induces frustrated autophagy in neurons. A, brain sections obtained from A␤PPswe-PS1de9 transgenic mice and control littermates were stained with p62 antibody. Nuclei were stained with Hoechst. Representative image of one of the brain sections with similar results in each case is shown. B, graphical representation of corrected total cell fluorescence of p62 in transgenic and wild-type brain sections. Difference in intensity of p62 staining is quantified by ImageJ as described under "Experimental Procedures." Data represent mean Ϯ S.E. of 30 different cells from three independent experiments. **, p Ͻ 0.001. C, cortical neurons were transfected with shTrib3 or shRand, and 48 h post-transfection they were treated with1.5 M A␤. p62 levels were determined by immunocytochemical staining using p62 antibody. D, percentage of stained cells indicates the proportions of transfected cells (green) with high (more or equal than the neighboring non-transfected cells) or low (less than the neighboring non transfected cells) p62 immunoreactivity levels after treatment with A␤. *, p Ͻ 0.05. E, PC12 cells were transfected with shTrib3 or shRand, primed, and then treated with 5 M A␤. Levels of p62 were analyzed by Western blotting using p62 antibody. F, graphical representation of fold change of p62 levels by densitometric analysis upon transfection with shTrib3 or shRand in presence or absence of A␤ 5 M. Data represents mean Ϯ S.E. of three independent experiments. *, p Ͻ 0.05. G, primed PC12 cells were transfected with Trib3 overexpression vector or PWPI as control. 48 h post-transfection cells were fixed, and p62 levels were checked by immunocytochemical staining using p62 antibody. H, graphical representation of corrected total cell fluorescence of p62 in differentiated PC12 cells transfected with PWPI or Trib3 OE vector. Difference in intensity of p62 staining is quantified by ImageJ as described under "Experimental Procedures." Data represent mean Ϯ S.E. of 30 different cells from three independent experiments. **, p Ͻ 0.001. (66 -69). mTOR is the master regulator of autophagy (52,70). The signaling cascade PI3K/Akt/mTOR is inhibited by A␤ (71). Trib3 inhibits the Akt/mTOR axis and leads to autophagy in human glioma cells (7). In corroboration with previous reports, we also found that A␤ induces dephosphorylation of mTOR and inactivates it. Down-regulating Trib3 blocked the dephosphorylation of mTOR after A␤ treatment. Moreover, we also observed that Ulk1, a direct target of mTOR, is activated upon A␤ treatment, which is required to initiate the autophagic cascade. Furthermore, we found an elevated level of the autophagosome marker LC3 in AD transgenic mice. Interestingly, down-regulating Trib3 blocked the activation of Ulk1 and demolished the increase in LC3 II upon A␤ treatment. This affirms the fact that Trib3 plays a critical role in A␤-induced autophagy. Furthermore, Trib3 regulates autophagy flux as well. We checked an important marker for autophagy flux, p62. In conditions of frustrated autophagy when the vacuoles are unable to fuse with the lysosomes, or when there is abnormal clearance of these vacuoles, there occurs accumulation of p62 within the cell (56). We observed that upon A␤ treatment there was an accumulation of p62; down-regulation of Trib3 abrogated this induction, and overexpressing Trib3 increases its accumulation. It has recently been reported that accumulation of p62 can mediate apoptosis via caspase-8 (72). However, accumulating evidence indicates that high levels of autophagy can lead to autosis, which is an autophagy-dependent non-apoptotic cell death (73,74). This suggests that Trib3 could also be involved in autophagic death induced by A␤ by increasing the aggregation of autophagic vacuoles with decreased clearance from the cells. The critical role of FoxO proteins in inducing autophagy in cardiomyocytes, hepatocytes, and skeletal muscles has also been well studied (75)(76)(77)(78). The exact mechanism by which Trib3 and FoxO together orchestrate autophagy in neurons still remains to be clearly elucidated.

Conclusions
Taken together, our results suggest a model in which Trib3 is involved in causing death of neurons treated with A␤ by a dual modality, whereby it induces apoptosis and also up-regulates autophagy with defective autophagic flux (Fig. 8C). Autophagic dysfunction is a multidimensional anomaly, identifying the exact nature of the defect is of utmost importance for therapeutic intervention (67). The signal that promotes apoptosis could also activate autophagy (79). Both apoptosis and autophagy are dysregulated in several neurodegenerative disorders, and there occurs an obscure cross-talk between the two phenomena (80,81). Our findings bring to light Trib3 as the molecule, which, via Akt, designs neuronal death paradigm by apoptosis and autophagy. We anticipate that Trib3 could act as a potential target for therapeutic intervention in AD.
Preparation of Amyloid-HPLC-purified A␤(1-42) was purchased from American Peptide (Sunnyvale, CA), and oligomeric A␤(1-42) was prepared. Lyophilized A␤(1-42) was reconstituted in HFIP to 1 mM; HFIP was then removed by evaporation in a SpeedVac, and it was then resuspended to a concentration of 5 mM in anhydrous DMSO. This stock was then stored in Ϫ80°C. The stock was further diluted with PBS to a final concentration of 400 M, and SDS was added to a final concentration of 0.2%. The resulting solution was incubated at 37°C for 18 -24 h. The preparation was finally diluted with PBS to a final concentration of 100 M and incubated at 37°C for 18 -24 h before use.
Oligomeric A␤ Treatment to Cells-Oligomeric A␤(1-42) was added to the medium containing cells for the specific time points. The concentrations used were 1.5 M for primary cultured rat cortical neurons and hippocampal neurons and 5 M for neuronally differentiated PC12 cells (38,44).
PCR-Total RNA for each sample is isolated from cultured cortical neurons by using TRI Reagent (Sigma). The primers used for PCR amplification of rat Trib3 were 5Ј-GTTGCGT-CGATTTGTCTTCA-3Ј and 5Ј-CGGGAGCTGAGTATCTC-TGG-3Ј. The primers for GAPDH were 5Ј-TCAACAGCAA-CTCCCACTCTT-3Ј and 5Ј-ACCCTGTTGCTGTAGCCG-TAT-3Ј. Equal amounts of cDNA template were used for each PCR analysis of Trib3 or GAPDH. Primers were used at 0.2 M concentration. For semi-quantitative PCR, products were analyzed on a 1.5% agarose gel and visualized by staining with ethidium bromide. Quantitative PCR were performed using One Step SYBR Ex Taq qRT-Takara by using Applied Biosystems 7500 Fast Real Time PCR System following the manufacturer's specifications.
Western Blotting Analysis-Cortical neurons and neuronally differentiated PC12 cells were lysed, and proteins were analyzed by Western blotting as described previously (65). For each condition 50 g of protein were resolved in 4 -12% SDS-PAGE and then transferred to PVDF membrane (Hybond: GE Healthcare, Buckinghamshire, UK). HRP-conjugated secondary antibodies against the primary antibodies were used. Detection was carried out by Amersham Biosciences ECL Western blotting detection reagent, according to the manufacturer's protocol. Imaging of all Western blottings was performed using an UVP BioImager 600 system equipped with Vision Works Life Science software, version 6.80 (UVP, Cambridge, UK).
Immunocytochemical Staining-Cortical neurons and neuronally differentiated PC12 cells were fixed with 4% paraformaldehyde for 10 min. Cells were then washed three times with PBS for 5 min each and then blocked in 3% goat serum in PBS containing 0.3% Triton X-100 for 2 h at room temperature. The cells were immunolabeled with primary antibody in a blocking solution overnight at 4 ºC. The next day, cells were washed in PBS, followed by incubation with the appropriate secondary antibody for 2 h at room temperature. Nuclei were stained with Hoechst. High resolution images were taken using a Leica TCS SP8 microscope (Germany). The intensities of staining for control or treated cells were quantified separately by ImageJ software. The corrected total cell fluorescence (CTCF) was determined by considering the integrated density of staining, area of the cell, and the background fluorescence for the different experimental conditions. CTCF ϭ integrated density Ϫ (area of selected cell ϫ mean fluorescence of background readings).
TUNEL Assay-Detection of TUNEL-positive cells using in situ cell death detection kit was performed following the manufacturer's protocol.
Mitochondrial Staining-Cellular mitochondria were detected using MitoTracker red dye following the manufacturer's protocol.
Immunoprecipitation-Primary cultured cortical neurons were either treated with 1.5 M A␤ for 8 h or left as untreated control, and interactions of Trib3 with Akt were detected by co-immunoprecipitation assays. The cells were washed with PBS and lysed in lysis buffer. For immunoprecipitation, agarose-conjugated anti-Trib3 antibody was prepared by incubating 3 g of anti-Trib3 antibody with 20 l of protein A-agarose beads for 2 h at 4 ºC under shaking conditions. The agaroseconjugated Trib3 antibodies were then pelleted down and incubated with treated and untreated cell lysates containing equal amounts of protein overnight. This was kept at 4 ºC under shaking condition. The antigen-antibody complexes were isolated and dissociated by boiling in sample buffer for 4 min. The agarose beads were pelleted down, and the supernatant was subjected to Western blotting analysis, as described previously using anti-Akt as the primary antibody.
Chromatin Immunoprecipitation (ChIP)-ChIP assays were done by using ChIP assay kit from Millipore (Billerica, MA) following manufacturer's protocol with few exceptions. 5-8 ϫ 10 6 cortical neurons were used after treatment with or without A␤. Rabbit polyclonal anti-FoxO1 antibody was used to immunoprecipitate the protein-DNA complexes. The primers used for PCR amplification of the rat Trib3 promoter were forward 5Ј-GTGCTGGGACTCCGAGATAG-3Ј and reverse 5Ј-CAACCTTCTTGCCAGACCTC-3Ј (23). PCR products were analyzed on a 1.5% agarose gel and visualized by staining with ethidium bromide.
Transfection-DNA was isolated using plasmid maxi kit from Qiagen. For survival assay, cortical, hippocampal neurons, and PC12 cells were transfected with 0.5 g of either pSIREN-Trib3-shRNA-zsgreen (shTrib3) or pSIREN-Rand-shRNAzsgreen (shRand), pWPI plasmid containing Trib3 overexpression vector, and the corresponding control plasmid. Transfections were done in 500 l of serum-free medium per well of a 24-well plate using Lipofectamine 2000. Five hours later, Lipofectamine-containing medium was replaced by fresh medium. Transfection was performed on the 3rd day of culture for primary cortical neurons and on the 19th day of culture for primary hippocampal neurons. Differentiated neuronal PC12 cells were transfected on the 3rd day of differentiation. For endogenous Trib3 down-regulation, naive PC12 cells were transfected with either 1 g of shTrib3 or shRand. Transfections were done in 1 ml of serum-free medium per well of a 12-well plate using Lipofectamine 2000. 24 h post-transfection, cells were differentiated in presence of NGF. After 5 days of priming, cells were treated with either A␤ (5 M) or left as untreated control.
Sholl Analysis-Sholl analysis was performed to analyze the neuritic branching and complexities of neuronal processes using ImageJ software as described previously (39) with a few modifications delineated below. Primary hippocampal neurons (19DIV) were transfected with shFoxO or shRand as described previously. A fluorescence microscope was used to image the transfected neurons. Low magnification pictures of single neurons were taken at 0 and 48 h of A␤(1-42) treatment. Sholl analysis was performed on these images using ImageJ software (Sholl analysis plugin). A number of concentric circles were drawn projecting from the cell body with gradually increasing radii of 40 mm in length. Two-dimensional analyses were performed to count the number of branches that intersect the successive concentric circle. Data are represented as mean Ϯ S.E. of four neurons from three independent experiments.
Survival Assay-Primary cortical neurons (3DIV) and primary hippocampal neurons (22DIV) were transfected with shRand or shTrib3 as mentioned above. 48 h post-transfection, the neurons were exposed to A␤ , and the number of transfected neurons (green) was counted (0 h) under the microscope. The number of surviving transfected neurons was also counted after 24, 48, and 72 h of treatment as described previously (49). Control and A␤-treated transfected neurons were imaged under a fluorescence microscope (Leica, Wetzlar, Germany). Data are represented as mean Ϯ S.E. of three independent experiments, which were performed in triplicate.
Oligomeric A␤ Infusion in Animals -Five Male Sprague-Dawley rats (300 -380 g) were infused with oligomeric A␤ as described previously, and five male rats were infused with PBS. Briefly, rats were anesthetized by injecting a mixture of xylazine/ketamine and placed on a stereotaxic frame. A volume of 5 l of 100 M A␤ in PBS was infused in the right cerebral cortex at stereotaxic coordinates from bregma: anterior-posterior, 4.1 mm, , 2.5 mm, dorsal-ventral, 1.3 mm, according to the rat brain atlas and a previous report (84). Equal volume of PBS was injected in control animals. Animals were sacrificed 21 days post-injection. The brains were dissected out following cardiac perfusion and fixed in 4% paraformaldehyde for 24 h. The brains were further incubated in a 30% sucrose solution for 24 h, and then cryo-sectioning was performed by using a cryotome (Thermo Fisher Scientific, West Palm Beach, FL).
ImmunohistochemistryofBrainSlices-Cryosectionsmeasuring 20 m of the brains from A␤-infused or PBS-infused rats and wild-type or transgenic mice were immunostained as described previously. Briefly, sections were blocked with 5% goat serum in PBS containing 0.3% Triton X-100 for 1 h at room temperature; sections were incubated in primary antibody in a blocking solution overnight at 4°C. The following day, the sections were washed three times with PBS and then incubated with a fluorescence-tagged secondary antibody for 2 h at room temperature. Hoechst staining for the nucleus was performed. The sections were mounted and observed under fluorescence microscope.
Statistics-All experimental results are reported as mean Ϯ S.E. Student's t test was performed as unpaired two-tailed sets of arrays to evaluate the significance of difference between the means and are presented as p values.
Author Contributions-S. C. B. and S. S. conceived the idea, designed the experiments, analyzed the data, and wrote the manuscript. S. S. performed all experiments.