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• W2567202920 abstract "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. 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) 2The abbreviations used are: ADAlzheimer's diseaseFoxOForkhead box, class OAββ-amyloidPC12pheochromocytoma cellsBimBcl-2 interacting mediator of cell deathmTORmammalian target of rapamycinLC3light chain 3ERendoplasmic reticulumDIVdays in vitroHFIP1,1,1,3,3,3-hexafluoro-2-propanol. 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 β-amyloid (Aβ) and neurofibrillary tangles of hyperphosphorylated Tau (1Serrano-Pozo A. Frosch M.P. Masliah E. Hyman B.T. Neuropathological alterations in Alzheimer disease.Cold Spring Harb. Perspect. Med. 2011; 1: a006189Crossref PubMed Scopus (1952) Google Scholar, 2Finder V.H. Glockshuber R. Amyloid-β aggregation.Neurodegener. Dis. 2007; 4: 13-27Crossref PubMed Scopus (261) Google Scholar). The disease is also characterized by accumulation of misfolded proteins, compromised autophagy, enhanced oxidative stress, and metabolic perturbations (3Tanzi R.E. Bertram L. Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective.Cell. 2005; 120: 545-555Abstract Full Text Full Text PDF PubMed Scopus (1492) Google Scholar, 4Selkoe D.J. Alzheimer's disease: genes, proteins, and therapy.Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5156) Google Scholar5LaFerla F.M. Green K.N. Oddo S. Intracellular amyloid-β in Alzheimer's disease.Nat. Rev. Neurosci. 2007; 8: 499-509Crossref PubMed Scopus (1567) Google Scholar). 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 (6Salminen A. Kauppinen A. Suuronen T. Kaarniranta K. Ojala J. ER stress in Alzheimer's disease: a novel neuronal trigger for inflammation and Alzheimer's pathology.J. Neuroinflammation. 2009; 6: 41Crossref PubMed Scopus (242) Google Scholar). In contrast, inappropriate aggregation of waste proteins and shoddy organelles induce autophagy via ER stress (7Salazar M. Carracedo A. Salanueva I.J. Hernández-Tiedra S. Lorente M. Egia A. Vázquez P. Blázquez C. Torres S. García S. Nowak J. Fimia G.M. Piacentini M. Cecconi F. Pandolfi P.P. et al.Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells.J. Clin. Invest. 2009; 119: 1359-1372Crossref PubMed Scopus (552) Google Scholar). Mounting evidence reveals the presence of accumulated autophagosomes and defective autophagy flux in the pathogenesis of AD (8François A. Rioux Bilan A. Quellard N. Fernandez B. Janet T. Chassaing D. Paccalin M. Terro F. Page G. Longitudinal follow-up of autophagy and inflammation in brain of APPswePS1dE9 transgenic mice.J. Neuroinflammation. 2014; 11: 139Crossref PubMed Scopus (59) Google Scholar, 9Nixon R.A. Mathews P.M. Cataldo A.M. The neuronal endosomal-lysosomal system in Alzheimer's disease.J. Alzheimers Dis. 2001; 3: 97-107Crossref PubMed Scopus (105) Google Scholar). It has also been demonstrated that Aβ 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 (10Ling D. Song H.J. Garza D. Neufeld T.P. Salvaterra P.M. Aβ42-induced neurodegeneration via an age-dependent autophagic-lysosomal injury in Drosophila.PLoS ONE. 2009; 4: e4201Crossref PubMed Scopus (136) Google Scholar11Hung S.Y. Huang W.P. Liou H.C. Fu W.M. Autophagy protects neuron from Aβ-induced cytotoxicity.Autophagy. 2009; 5: 502-510Crossref PubMed Scopus (170) Google Scholar, 12Jaeger P.A. Wyss-Coray T. All-you-can-eat: autophagy in neurodegeneration and neuroprotection.Mol. Neurodegener. 2009; 4: 16Crossref PubMed Scopus (140) Google Scholar13Boland B. Kumar A. Lee S. Platt F.M. Wegiel J. Yu W.H. Nixon R.A. Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease.J. Neurosci. 2008; 28: 6926-6937Crossref PubMed Scopus (862) Google Scholar). Alzheimer's disease Forkhead box, class O β-amyloid pheochromocytoma cells Bcl-2 interacting mediator of cell death mammalian target of rapamycin light chain 3 endoplasmic reticulum days in vitro 1,1,1,3,3,3-hexafluoro-2-propanol. 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 (14Salazar M. Carracedo A. Salanueva I.J. Hernández-Tiedra S. Egia A. Lorente M. Vázquez P. Torres S. Iovanna J.L. Guzmán M. Boya P. Velasco G. TRB3 links ER stress to autophagy in cannabinoid anti-tumoral action.Autophagy. 2009; 5: 1048-1049Crossref PubMed Scopus (60) Google Scholar, 15Vara D. Salazar M. Olea-Herrero N. Guzmán M. Velasco G. Díaz-Laviada I. Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role of AMPK-dependent activation of autophagy.Cell Death Differ. 2011; 18: 1099-1111Crossref PubMed Scopus (203) Google Scholar). Trib3 is a mammalian ortholog of the Drosophila Tribbles gene and is also known as neuronal death-inducible putative kinase/Sink1/Skip3 (16Ord D. Ord T. Characterization of human NIPK (TRB3, SKIP3) gene activation in stressful conditions.Biochem. Biophys. Res. Commun. 2005; 330: 210-218Crossref PubMed Scopus (88) Google Scholar). Trib3 is responsible for a plethora of functions ranging from glucose regulation, migration of tumor cells, suppressing differentiation of adipocytes, and cell cycle control (17Hua F. Mu R. Liu J. Xue J. Wang Z. Lin H. Yang H. Chen X. Hu Z. TRB3 interacts with SMAD3 promoting tumor cell migration and invasion.J. Cell Sci. 2011; 124: 3235-3246Crossref PubMed Scopus (109) Google Scholar18Ord D. Meerits K. Ord T. TRB3 protects cells against the growth inhibitory and cytotoxic effect of ATF4.Exp. Cell Res. 2007; 313: 3556-3567Crossref PubMed Scopus (72) Google Scholar, 19Takahashi Y. Ohoka N. Hayashi H. Sato R. TRB3 suppresses adipocyte differentiation by negatively regulating PPARγ transcriptional activity.J. Lipid Res. 2008; 49: 880-892Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar20Sakai S. Ohoka N. Onozaki K. Kitagawa M. Nakanishi M. Hayashi H. Dual mode of regulation of cell division cycle 25 A protein by TRB3.Biol. Pharm. Bull. 2010; 33: 1112-1116Crossref PubMed Scopus (13) Google Scholar). It was identified as a novel ER stress-inducible gene that, when up-regulated, activated several genes involved in cell death during ER stress (21Ohoka N. Yoshii S. Hattori T. Onozaki K. Hayashi H. TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death.EMBO J. 2005; 24: 1243-1255Crossref PubMed Scopus (749) Google Scholar). Trib3 is also shown to be elevated by several stresses, including hypoxia, 6-hydroxydopamine, growth factor deprivation, anoxia, and ethanol exposure (16Ord D. Ord T. Characterization of human NIPK (TRB3, SKIP3) gene activation in stressful conditions.Biochem. Biophys. Res. Commun. 2005; 330: 210-218Crossref PubMed Scopus (88) Google Scholar, 22Wennemers M. Bussink J. Scheijen B. Nagtegaal I.D. van Laarhoven H.W. Raleigh J.A. Varia M.A. Heuvel J.J. Rouschop K.M. Sweep F.C. Span P.N. Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response.Breast Cancer Res. 2011; 13: R82Crossref PubMed Scopus (71) Google Scholar23Zareen N. Biswas S.C. Greene L.A. A feed-forward loop involving Trib3, Akt and FoxO mediates death of NGF-deprived neurons.Cell Death Differ. 2013; 20: 1719-1730Crossref PubMed Scopus (40) Google Scholar, 24Corcoran C.A. Luo X. He Q. Jiang C. Huang Y. Sheikh M.S. Genotoxic and endoplasmic reticulum stresses differentially regulate TRB3 expression.Cancer Biol. Ther. 2005; 4: 1063-1067Crossref PubMed Scopus (48) Google Scholar, 25Wu M. Xu L.G. Zhai Z. Shu H.B. SINK is a p65-interacting negative regulator of NF-κB-dependent transcription.J. Biol. Chem. 2003; 278: 27072-27079Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Zhang J. Wen H.J. Guo Z.M. Zeng M.S. Li M.Z. Jiang Y.E. He X.G. Sun C.Z. TRB3 overexpression due to endoplasmic reticulum stress inhibits AKT kinase activation of tongue squamous cell carcinoma.Oral Oncol. 2011; 47: 934-939Crossref PubMed Scopus (35) Google Scholar, 27Aimé P. Sun X. Zareen N. Rao A. Berman Z. Volpicelli-Daley L. Bernd P. Crary J.F. Levy O.A. Greene L.A. Trib3 is elevated in Parkinson's disease and mediates death in Parkinson's disease models.J. Neurosci. 2015; 35: 10731-10749Crossref PubMed Scopus (39) Google Scholar28Zou C.G. Cao X.Z. Zhao Y.S. Gao S.Y. Li S.D. Liu X.Y. Zhang Y. Zhang K.Q. The molecular mechanism of endoplasmic reticulum stress-induced apoptosis in PC-12 neuronal cells: the protective effect of insulin-like growth factor I.Endocrinology. 2009; 150: 277-285Crossref PubMed Scopus (74) Google Scholar). It has also been shown that Trib3 is elevated in Parkinson's disease brains and mediates neuron death in various Parkinson's disease models (27Aimé P. Sun X. Zareen N. Rao A. Berman Z. Volpicelli-Daley L. Bernd P. Crary J.F. Levy O.A. Greene L.A. Trib3 is elevated in Parkinson's disease and mediates death in Parkinson's disease models.J. Neurosci. 2015; 35: 10731-10749Crossref PubMed Scopus (39) Google Scholar). Trib3 is a pseudokinase because it lacks the catalytic residues required for its kinase function (29Boudeau J. Miranda-Saavedra D. Barton G.J. Alessi D.R. Emerging roles of pseudokinases.Trends Cell Biol. 2006; 16: 443-452Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 30Kiss-Toth E. Bagstaff S.M. Sung H.Y. Jozsa V. Dempsey C. Caunt J.C. Oxley K.M. Wyllie D.H. Polgar T. Harte M. O'Neill L.A. Qwarnstrom E.E. Dower S.K. Human tribbles, a protein family controlling mitogen-activated protein kinase cascades.J. Biol. Chem. 2004; 279: 42703-42708Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). 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 (25Wu M. Xu L.G. Zhai Z. Shu H.B. SINK is a p65-interacting negative regulator of NF-κB-dependent transcription.J. Biol. Chem. 2003; 278: 27072-27079Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 31Hegedus Z. Czibula A. Kiss-Toth E. Tribbles: novel regulators of cell function; evolutionary aspects.Cell. Mol. Life Sci. 2006; 63: 1632-1641Crossref PubMed Scopus (73) Google Scholar, 32Liew C.W. Bochenski J. Kawamori D. Hu J. Leech C.A. Wanic K. Malecki M. Warram J.H. Qi L. Krolewski A.S. Kulkarni R.N. The pseudokinase tribbles homolog 3 interacts with ATF4 to negatively regulate insulin exocytosis in human and mouse beta cells.J. Clin. Invest. 2010; 120: 2876-2888Crossref PubMed Scopus (99) Google Scholar33Qi L. Heredia J.E. Altarejos J.Y. Screaton R. Goebel N. Niessen S. Macleod I.X. Liew C.W. Kulkarni R.N. Bain J. Newgard C. Nelson M. Evans R.M. Yates J. Montminy M. TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism.Science. 2006; 312: 1763-1766Crossref PubMed Scopus (248) Google Scholar). 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 Aβ cascade hypothesis holds the spotlight of the pathogenesis of the disease, targeting Aβ proves to be a promising approach (34Huang Y. Mucke L. Alzheimer mechanisms and therapeutic strategies.Cell. 2012; 148: 1204-1222Abstract Full Text Full Text PDF PubMed Scopus (1358) Google Scholar, 35Musiek E.S. Holtzman D.M. Three dimensions of the amyloid hypothesis: time, space and 'wingmen'.Nat. Neurosci. 2015; 18: 800-806Crossref PubMed Scopus (468) Google Scholar). Apart from this, a complementary therapy is imperative to impede the toxicity due to Aβ, the complete removal of which is difficult. Hence, a complete understanding of the molecular mechanism of Aβ-induced death is quintessential. In this study, we have investigated the role of Trib3 in neuronal death induced by Aβ. It appears that Trib3 is induced and promotes death of neurons by both apoptosis and autophagy in response to Aβ. Accumulating evidence implicates Aβ oligomers as the principal cause of AD pathogenesis (36Gilbert B.J. The role of amyloid β in the pathogenesis of Alzheimer's disease.J. Clin. Pathol. 2013; 66: 362-366Crossref PubMed Scopus (102) Google Scholar, 37Lesné S.E. Sherman M.A. Grant M. Kuskowski M. Schneider J.A. Bennett D.A. Ashe K.H. Brain amyloid-β oligomers in ageing and Alzheimer's disease.Brain. 2013; 136: 1383-1398Crossref PubMed Scopus (335) Google Scholar). Oligomeric Aβ at a concentration of 1.5 μm leads to significant death of primary cortical and hippocampal neurons after 24 h of exposure (38Sanphui P. Biswas S.C. FoxO3a is activated and executes neuron death via Bim in response to β-amyloid.Cell Death Dis. 2013; 4: e625Crossref PubMed Scopus (96) Google Scholar). We determined the levels of Trib3 in neurons after Aβ exposure. We found that Trib3 levels were increased in cultured cortical neurons following Aβ(1–42) treatment. To check the specificity of the action of Aβ(1–42), we used a reverse peptide, Aβ(42–1), and we found that the reverse peptide Aβ(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 Aβ(1–42) treatment as detected by semi-quantitative (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 Aβ treatment, respectively (Fig. 1, C and D). Thus, Trib3 expression was elevated well before cell death became apparent (see below and Fig. 2C).FIGURE 2Trib3 is essential for neuronal death evoked by Aβ. A, PC12 cells were transfected with shTrib3 or shRand, primed, and then treated with and without Aβ (5 μm). Levels of endogenous Trib3 levels were assessed by Western blotting analysis using anti-Trib3 antibody. Lane C, control. B, graphical representation of fold change of Trib3 levels by densitometric analysis of Western blottings. Data represent mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.005. Primary cultured rat cortical neurons (5DIV) (C) and primary cultured hippocampal neurons (19DIV) (E) were transfected with pSIREN-shTrib3-zsgreen (shTrib3) or control pSIREN-shRand-zsgreen (shRand) and maintained for 48 h and then subjected to Aβ (1.5 μm) treatment for 72 h. Representative pictures of transfected neurons that were maintained in the presence or absence of Aβ 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 Aβ 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 ± S.E., performed in triplicate. The asterisks denote statistically significant differences from control (shRand) at corresponding time points: *, p < 0.05; **, p < 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 ± S.E. of six different neurons from three independent cultures for each class. Asterisks denote statistically significant differences from shRand (control): *, p < 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 μm Aβ for 16 h, after which they were immunostained with MitoTracker red dye (H), PSD95 (I), or cleaved caspase3 antibodies (J).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we investigated whether this increase of Trib3 in vitro is also reflected in in vivo conditions. Reports reveal that oligomeric Aβ when infused into adult rat brains results in Aβ deposition, caspase-3 activation, and neuronal cell loss in the vicinity of Aβ infusion (38Sanphui P. Biswas S.C. FoxO3a is activated and executes neuron death via Bim in response to β-amyloid.Cell Death Dis. 2013; 4: e625Crossref PubMed Scopus (96) Google Scholar). In our study adult rats were infused with Aβ 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 Aβ(1–42) antibody to check Aβ deposition in the site of Aβ 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 Aβ-infused rat brains as compared with PBS-infused rat brains (Fig. 1, F and G). Because synthetic Aβ may behave differently from naturally secreted Aβ, brain sections of APPswe-PS1de9 (Swedish mutation in amyloid precursor protein and PS1 mutation) transgenic mice, which naturally secrete Aβ, were also examined for Trib3 expression. The presence of Aβ 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 Aβ in vitro and in vivo. To find the role of Trib3 in Aβ-induced neuronal cell death in vivo, we performed immunostaining of Trib3 along with TUNEL assay in sections in the vicinity of Aβ infusion. We observed that increased Trib3 expression co-localized with TUNEL-positive cells in Aβ-infused rat brain sections as compared with PBS-infused rats (Fig. 1K, see arrows). This indicates the role of Trib3 in Aβ-induced neuronal cell death in vivo. We next assessed whether Trib3 is necessary in evoking neuronal death in response to Aβ. We interfered with the expression of Trib3 using previously described shRNA constructs (23Zareen N. Biswas S.C. Greene L.A. A feed-forward loop involving Trib3, Akt and FoxO mediates death of NGF-deprived neurons.Cell Death Differ. 2013; 20: 1719-1730Crossref PubMed Scopus (40) Google Scholar). The Trib3 shRNA construct efficiently blocked induction of Trib3 by Aβ 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 Aβ treatment (1.5 μm). 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 Aβ 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 Aβ-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 Aβ treatment by Sholl analysis as described (38Sanphui P. Biswas S.C. FoxO3a is activated and executes neuron death via Bim in response to β-amyloid.Cell Death Dis. 2013; 4: e625Crossref PubMed Scopus (96) Google Scholar, 39Cuesto G. Enriquez-Barreto L. Caramés C. Cantarero M. Gasull X. Sandi C. Ferrús A. Acebes Á. Morales M. Phosphoinositide-3-kinase activation controls synaptogenesis and spinogenesis in hippocampal neurons.J. Neurosci. 2011; 31: 2721-2733Crossref PubMed Scopus (107) Google Scholar). 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 Aβ 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 Aβ-treated conditions. We also found a similar protective effect of shTrib3 on neuronal PC12 cells upon Aβ treatment (data not shown). Taken together, these results suggest that Trib3 plays a necessary role in mediating neuron degeneration and death evoked by Aβ toxicity. It has been shown that Trib3 blocks Akt activity by physically interacting with it in various non-neuronal cells (40He L. Simmen F.A. Mehendale H.M. Ronis M.J. Badger T.M. Chronic ethanol intake impairs insulin signaling in rats by disrupting Akt association with the cell membrane. Role of TRB3 in inhibition of Akt/protein kinase B activation.J. Biol. Chem. 2006; 281: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 41Cravero J.D. Carlson C.S. Im H.J. Yammani R.R. Long D. Loeser R.F. Increased expression of the Akt/PKB inhibitor TRB3 in osteoarthritic chondrocytes inhibits insulin-like growth factor 1-mediated cell survival and proteoglycan synthesis.Arthritis Rheum. 2009; 60: 492-500Crossref PubMed Scopus (68) Google Scholar42Du K. Herzig S. Kulkarni R.N. Montminy M. TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver.Science. 2003; 300: 1574-1577Crossref PubMed Scopus (709) Google Scholar). As phospho-Akt level is reported to be markedly reduced in AD brain (43Lee H.K. Kumar P. Fu Q. Rosen K.M. Querfurth H.W. The insulin/Akt signaling pathway is targeted by intracellular β-amyloid.Mol. Biol. Cell. 2009; 20: 1533-1544Crossref PubMed Scopus (176) Google Scholar), we tested whether Trib3 also negatively regulates Akt in neuronal cells in response to Aβ. Primary cultures of cortical neurons were treated with Aβ, 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 Aβ-treated neurons compared with control neurons (Fig. 3, A and B). We then checked the phosphorylation status of Akt upon Aβ treatment. We observed that the decrease in phosphorylation at Ser-473 of Akt caused by Aβ 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 Aβ, the protein levels of Trib3 increased even in the absence of Aβ (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 nucleus and induces its target genes in Aβ-treated neurons (38Sanphui P. Biswas S.C. FoxO3a is activated and executes neuron death via Bim in response to β-amyloid.Cell Death Dis. 2013; 4: e625Crossref PubMed Scopus (96) Google Scholar, 44Akhter R. Sanphui P. Biswas S.C. The essential role of p53-up-regulated modulator of apoptosis (Puma) and its regulation by FoxO3a transcription factor in β-amyloid-induced neuron death.J. Biol. Chem. 2014; 289: 10812-10822Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Previous reports reveal the presence of putative FoxO-binding sites on the Trib3 promoter (45Furuyama T. Nakazawa T. Nakano I. Mori N. Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues.Biochem. J. 2000; 349: 629-634Crossref PubMed Scopus (549) Google Scholar46Sandelin A. Alkema W. Engström P. Wasserman W.W. Lenhard B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles.Nucleic Acids Res. 2004; 32: D91-D94Crossref PubMed Google Scholar, 47Farré D. Roset R. Huerta M. Adsuara J.E. Roselló L. Albà M.M. Messeguer X. Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN.Nucleic Acids Res. 2003; 31: 3651-3653Crossref PubMed Scopus (752) Google Scholar48Xuan Z. Zhang M.Q. From worm to human: bioinformatics approaches to identify FOXO target genes.Mech. Ageing Dev. 2005; 126: 209-215Crossref PubMed Scopus (49) Google Scholar). We therefore hypothesized that Trib3 may be regulated by FoxO transcription factors in Aβ-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 Aβ (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 Aβ treatment. Previous results prompted us to test whether" @default.
• W2567202920 created "2017-01-06" @default.
• W2567202920 creator A5047102920 @default.
• W2567202920 creator A5058730974 @default.
• W2567202920 date "2017-02-01" @default.
• W2567202920 modified "2023-10-16" @default.
• W2567202920 title "Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-β-induced Neuronal Death" @default.
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