Vicious Cycle? Aβ Triggers Cell Stress, Generating More Aβ

Cellular stress has been implicated in many neurodegenerative diseases, but little is known about how its pathways interact with pathology. Now, in the September 6 Neuron, researchers led by Sung Yoon and Kun Huang at The Ohio State University, Columbus, report that Aβ oligomers can activate metabolic stress pathways by blocking protein translation. The increased stress turns on a kinase involved in cell death, JNK3, which then acts to ramp up production of Aβ, the authors report. This harmful feedback loop might make JNK3 a therapeutic target, Yoon proposed. “Our data strongly suggest a positive feed-forward mechanism that is perpetuated by JNK3, so if we halt it in some manner, maybe we could significantly lower [Aβ levels],” she told Alzforum.

Cellular stressors, such as hoards of misfolded proteins, trigger the unfolded protein response (UPR), a coping mechanism that switches on early in conditions such as amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease (see ARF related news story). As part of this response, cells activate JNK (see Urano et al., 2000). Previous work by Yoon and others showed that the brain-specific isoform JNK3 mediates neuronal death in Parkinson’s disease (see Hunot et al., 2004) and Huntington’s disease (see ARF related news story), as well as the death of neurons and oligodendrocytes after brain injury (see Beffert et al., 2006; Li et al., 2007). “The new study is thorough and presents compelling evidence that Aβ can play a role in activating JNK3,” said Uwe Beffert, Boston University, Massachusetts. Beffert has collaborated with Yoon in the past but was not involved in the current research.

The route from Aβ to the kinase is an indirect one, however. Yoon and colleagues first showed that the peptides suppress the translational machinery that synthesizes proteins from messenger RNA. In rat hippocampal neuron cultures treated overnight with synthetic Aβ42 peptides (5 μM), protein synthesis dropped by about half. The authors traced the mechanism to the activation of AMP-activated protein kinase α (AMPKα), which is known to block initiation of translation (see Gwinn et al., 2008; Inoki et al., 2003). Because translation sputters, it kicks off the unfolded protein response and activation of JNK3, the authors report. How Aβ might activate AMPKα is not clear, but the authors speculate the peptide may pump up calcium levels, thereby activating CaMK, which can phosphorylate AMPKα. Intriguingly, Aβ has been shown to flood cells with calcium from internal stores, triggering the UPR (see ARF related news story).

What form of Aβ is responsible for blocking translation? Western blots of the peptide solution showed that most of it was monomeric, with about 5 percent being small oligomers, mostly dimers and trimers, the authors report. Control experiments showed that fibrillar and pure monomeric Aβ did not activate AMPKα, implying that oligomers were the active species in these experiments. Their concentration in the mixture was about 250 nM, which is higher than some estimates of physiological concentrations of Aβ42 in AD brains. The field has grappled with the issue of what is the physiologically relevant form and concentration of Aβ (see ARF Webinar).

Yoon and colleagues then turned to in-vivo studies. They crossed 5xFAD transgenic mice with JNK3 knockout mice, and saw dramatic improvements in pathology. At one year old, the 5xFAD/JNK3 knockouts accumulated about one-quarter the amount of insoluble Aβ in multiple brain regions as did their JNK3-positive cousins. Fewer cortical neurons died in the absence of JNK3 than in transgenic controls, although still more than in wild-type animals. Memory in a fear paradigm approached wild-type levels in the crosses. This suggests a role for JNK3 in promoting AD-like pathology and neuronal damage in mice.

The authors also examined where JNK3 is expressed and what it does in 5xFAD transgenics. Immunohistochemistry showed that phosphorylated JNK3 loiters near amyloid plaques and dystrophic neurites in 5x mice, Tg2576 mice, and aged monkeys. They found that at six months of age, JNK3 is about 25 percent more active in transgenics than in wild-type mice, an increase similar to what is seen in human postmortem AD brains. Both findings jibe with previous reports that JNK3 switches on in damaged neurites (see, e.g., Muresan and Muresan, 2005; Cavalli et al., 2005), and places the kinase in a relevant location to play a role in AD.

Finally, the authors linked the kinase to altered processing of amyloid precursor protein (APP). In cell culture experiments, JNK3 phosphorylated APP at position T668, as shown previously by other groups. Treating cultures with JNK activators rapidly increased APP at the cell surface, followed by a gradual reuptake that cut the amount of surface-bound APP in half within 30 minutes. This implied to the authors that JNK3 facilitates the internalization of APP. Since amyloidogenic processing is believed to occur largely in endosomal compartments, this would pump up Aβ levels, the authors note. Mutation of the T668 phosphorylation site, or treatment with a JNK inhibitor, prevented the response.

JNK3-negative 5xFAD mice displayed less phosphorylated APP at the cell membrane than normal 5x animals, but had normal APP phosphorylation in whole-cell lysates. The authors suggest that JNK3 preferentially affects membrane-bound APP. However, Beffert noted that the change in phosphorylated APP is small, and questioned whether it could lead to the large changes seen in insoluble Aβ levels. JNK3 may be acting on other substrates that affect amyloid processing as well, he suggested.

Yoon believes that JNK3 could make a therapeutic target. She plans to test inhibitors in several strains of AD mice to see if they lower Aβ levels and improve symptoms, particularly when given late in the disease course.—Madolyn Bowman Rogers

Comments

  1. The mechanisms that lead to the pathology of Alzheimer’s disease (AD) are not completely understood. However, numerous hypotheses have been proposed to explain the main pathological features characteristic for the AD brain—among these, the increased production and accumulation of toxic amyloid-β (Aβ) species. Some of these hypotheses appear to hold when tested in cell culture, in animal models of AD, as well as when confronted with the real AD brain.

    In their recent study, Yoon et al. came up with just such a hypothesis. They identify a pathway that could explain how an initial accumulation of oligomeric Aβ—probably caused by random fluctuations in the cell metabolism—could trigger a self-amplifying loop that produces and accumulates more toxic Aβ species. Briefly, the authors propose that the trigger of this pathway is a block of translation caused by an initial increase in extracellular Aβ oligomers. This is a stress, sensed by one of the neuron’s stress centers, the endoplasmic reticulum (ER), which unleashes a typical unfolded protein response (UPR) that activates the stress kinase, c-Jun N-terminal kinase 3 (JNK3). JNK3 then phosphorylates specific substrates—among them the Aβ precursor protein (APP). The phosphorylated APP (pAPP) is subjected to increased processing via the amyloidogenic pathway—a fact already known from previous studies (1)—likely through increased routing from the cell surface into endosomes, which provide a friendly, i.e., acidic, environment for secretase cleavage. In this way, a small, initial amount of oligomeric Aβ amplifies the generation and accumulation of toxic Aβ species. The authors' data also provide support for an interesting and logical pathway—involving activation of the kinase AMPK and inhibition of mTOR—for the initial events leading to the Aβ-triggered translational block.

    Here, we would like to draw attention to two facts that appear essential for the increased production of Aβ, and which actually go hand in hand: the ER stress response and the phosphorylation of APP by JNK. In recent years, numerous studies (including ours) have pointed to stress—often mediated by an ER response—as the cause for neuronal degeneration and death in neurodegenerative diseases, including AD (2-12). Second, while it was known for some time that pAPP is a better substrate for amyloidogenic processing than non-phosphorylated APP (1), the fact that the phosphorylation of APP at Thr668 (numbering for the APP695 neuronal isoform) could be an obligatory event for the development of robust, AD-specific pathology is a finding of recent studies, including the one discussed here (1,14-16). These more recent studies (including one from our laboratory) also point to the role played by JNK—more specifically, JNK3—in the phosphorylation of Thr668 of APP and, likely, in the pathogenic process in AD (10,13,15). However, it has to be noted that the same residue (Thr668) can be—and actually is, under certain circumstances—phosphorylated by other kinases, including cyclin-dependent kinase 5 (Cdk5) (1,16,17). In this respect, of particular interest is the fact that a mouse model of AD-type neurodegeneration that reproduces fairly well both the amyloid and tau pathology is one that exogenously expresses p25, an activator of Cdk5 (18,19). We have also shown in cell culture that activation of Cdk5 leads to hyperphosphorylation of APP and neurodegeneration (17).

    Finally, we would like to point out that—almost as a rule—it appears that the response to a cellular stress, rather than the stress itself, generates the toxic Aβ species, which accumulate and oligomerize. The initial stress may vary; it can be an oxidative stress, a block of translation, or an impeded axonal transport, as we recently proposed (10). In our recently published model, the block of transport leads to the accumulation of APP and recruitment to APP of the active JNK complex in neuronal soma at the ER. This leads to the phosphorylation of APP, followed by its amyloidogenic processing and the release of Aβ in the ER lumen, where it accumulates and oligomerizes. Certainly, there are many stress-activated pathways that can trigger abnormal production and accumulation of toxic Aβ species, and they all, in principle, could affect the metabolism of APP, and—if stress is sustained—could lead to AD pathology. For now, it appears that a number of these pathways involve an ER response to a stress, and lead to the phosphorylation of APP.

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    Bernard-Marissal N, Moumen A, Sunyach C, Pellegrino C, Dudley K, Henderson CE, Raoul C, Pettmann B. Reduced calreticulin levels link endoplasmic reticulum stress and Fas-triggered cell death in motoneurons vulnerable to ALS. J Neurosci. 2012 Apr 4;32(14):4901-12. PubMed.

    Carroll JC, Iba M, Bangasser DA, Valentino RJ, James MJ, Brunden KR, Lee VM, Trojanowski JQ. Chronic stress exacerbates tau pathology, neurodegeneration, and cognitive performance through a corticotropin-releasing factor receptor-dependent mechanism in a transgenic mouse model of tauopathy. J Neurosci. 2011 Oct 5;31(40):14436-49. PubMed.

    Colla E, Coune P, Liu Y, Pletnikova O, Troncoso JC, Iwatsubo T, Schneider BL, Lee MK. Endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3306-20. PubMed.

    Colla E, Jensen PH, Pletnikova O, Troncoso JC, Glabe C, Lee MK. Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo. J Neurosci. 2012 Mar 7;32(10):3301-5. PubMed.

    Ferreiro E, Baldeiras I, Ferreira IL, Costa RO, Rego AC, Pereira CF, Oliveira CR. Mitochondrial- and endoplasmic reticulum-associated oxidative stress in Alzheimer's disease: from pathogenesis to biomarkers. Int J Cell Biol. 2012;2012:735206. PubMed.

    Ghosh D, Levault KR, Barnett AJ, Brewer GJ. A reversible early oxidized redox state that precedes macromolecular ROS damage in aging nontransgenic and 3xTg-AD mouse neurons. J Neurosci. 2012 Apr 25;32(17):5821-32. PubMed.

    Kang MJ, Chung J, Ryoo HD. CDK5 and MEKK1 mediate pro-apoptotic signalling following endoplasmic reticulum stress in an autosomal dominant retinitis pigmentosa model. Nat Cell Biol. 2012 Apr;14(4):409-15. PubMed.

    Mendonsa G, Dobrowolska J, Lin A, Vijairania P, Jong YJ, Baenziger NL. Molecular profiling reveals diversity of stress signal transduction cascades in highly penetrant Alzheimer's disease human skin fibroblasts. PLoS One. 2009;4(2):e4655. PubMed.

    Muresan V, Muresan Z. A persistent stress response to impeded axonal transport leads to accumulation of amyloid-β in the endoplasmic reticulum, and is a probable cause of sporadic Alzheimer's disease. Neurodegener Dis. 2012;10(1-4):60-3. PubMed.

    Ohta K, Mizuno A, Li S, Itoh M, Ueda M, Ohta E, Hida Y, Wang MX, Furoi M, Tsuzuki Y, Sobajima M, Bohmoto Y, Fukushima T, Kobori M, Inuzuka T, Nakagawa T. Endoplasmic reticulum stress enhances γ-secretase activity. Biochem Biophys Res Commun. 2011 Dec 16;416(3-4):362-6. PubMed.

    Wang S, Kaufman RJ. The impact of the unfolded protein response on human disease. J Cell Biol. 2012 Jun 25;197(7):857-67. PubMed.

    Mazzitelli S, Xu P, Ferrer I, Davis RJ, Tournier C. The loss of c-Jun N-terminal protein kinase activity prevents the amyloidogenic cleavage of amyloid precursor protein and the formation of amyloid plaques in vivo. J Neurosci. 2011 Nov 23;31(47):16969-76. PubMed.

    Oulès B, Del Prete D, Greco B, Zhang X, Lauritzen I, Sevalle J, Moreno S, Paterlini-Bréchot P, Trebak M, Checler F, Benfenati F, Chami M. Ryanodine receptor blockade reduces amyloid-β load and memory impairments in Tg2576 mouse model of Alzheimer disease. J Neurosci. 2012 Aug 22;32(34):11820-34. PubMed.

    Sclip A, Antoniou X, Colombo A, Camici GG, Pozzi L, Cardinetti D, Feligioni M, Veglianese P, Bahlmann FH, Cervo L, Balducci C, Costa C, Tozzi A, Calabresi P, Forloni G, Borsello T. c-Jun N-terminal kinase regulates soluble Aβ oligomers and cognitive impairment in AD mouse model. J Biol Chem. 2011 Dec 23;286(51):43871-80. PubMed.

    Iijima K, Ando K, Takeda S, Satoh Y, Seki T, Itohara S, Greengard P, Kirino Y, Nairn AC, Suzuki T. Neuron-specific phosphorylation of Alzheimer's beta-amyloid precursor protein by cyclin-dependent kinase 5. J Neurochem. 2000 Sep;75(3):1085-91. PubMed.

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    View all comments by Virgil Muresan

  2. Under diverse stress conditions, such as a perturbed calcium homeostasis, the normal function of the endoplasmic reticulum (ER) is impaired, leading to a phenomenon known as ER stress. To reestablish homeostasis and normal ER function, mammalian cells evolved a coordinated response of protein signaling pathways and transcription factors termed the unfolded protein response (UPR). This adaptive response initiates ER-to-nucleus signaling cascades that involve the transcriptional upregulation of genes that increase the ER folding capacity, protein quality control, and degradation of terminally misfolded proteins. In addition, the influx of newly synthesized proteins into the ER is reduced through induction of general translational arrest. This reduction in the global rate of translation is one of the earliest events in the UPR, and it was reported to inhibit long-term potentiation and memory acquisition. Accordingly, recent observations suggest that deregulation of the UPR, or chronic ER stress, is a fundamental pathological event in many neurodegenerative disorders, such as Alzheimer’s disease (AD).

    ER stress markers have been found in postmortem samples from patients affected with AD, as well in cellular and animal models of this brain disorder. Pharmacological strategies and genetic manipulation in cellular and animal disease models have provided promising results concerning the contribution of ER stress to the neurodegenerative process. Several in-vitro studies support the finding that UPR activation and ER stress are induced by the amyloid-β (Aβ) peptide. We found that Aβ oligomers, which have been suggested to be the main neurotoxins in AD, impair ER calcium homeostasis and activate the UPR, leading to an ER stress-mediated apoptotic cell death pathway in cortical and hippocampal neurons (Resende et al., 2008; Costa et al., 2012). Yoon and colleagues now provide clear evidence that Aβ oligomers block translation, leading to widespread ER stress, and they clarified the molecular mechanisms implicated in these events. Through an elegant and well-designed study that used several cellular and animal models, as well as AD brain samples, the authors demonstrated that oligomeric Aβ-induced translational block and subsequent ER stress activates JNK3, which in turn phosphorylates APP, facilitating its endocytosis and subsequent amyloidogenic processing and Aβ42 production. In a familial AD mouse model, Yoon et al. further demonstrated that JNK3 deletion reduces Aβ42 levels and plaque loads, increases neuronal numbers, and improves cognition, thus implicating ER stress-mediated JNK3 activation in the neurodegenerative process and cognitive deficits induced by Aβ oligomers in AD.

    Yoon and colleagues found that AMP-activated protein kinase (AMPK) activation and subsequent mammalian target of rapamycin inhibition occur upstream of Aβ-induced translational block and subsequent ER stress, showing that perturbation of energy metabolism plays a major causal role in the synaptotoxic effect of Aβ oligomers, and supporting the idea that metabolic deficits have a major role during preclinical AD. Indeed, brain imaging studies demonstrated that cerebral glucose utilization is reduced in the brains of AD patients and, more importantly, in mild cognitive impairment (MCI) subjects. These metabolic alterations that occur in the initial stages of the pathology seem to arise from the deleterious effect of Aβ in mitochondria. Aβ has been shown to accumulate in the mitochondria (resulting, at least in part, from APP processing within this organelle) and to induce structural and functional alterations that culminate in ATP depletion, oxidative stress, and activation of cell death pathways.

    Based on the present findings, the authors propose that a plausible explanation for AMPK activation by Aβ oligomers is the perturbation of intracellular calcium homeostasis and subsequent activation of kinases such as calmodulin kinase. This hypothesis is supported by recent studies from our lab demonstrating that ER stress occurs downstream of activation of the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR) and perturbation of intracellular calcium levels in neuronal cultures treated with Aβ oligomers (Costa et al., 2012; Ferreira et al., 2012). The paper by Yoon et al. suggests that Aβ oligomers trigger a positive feedback loop during the initial stages of the disease that culminates in synaptic dysfunction and cognitive deficits. As demonstrated in the paper, oligomeric Aβ induces translational block and ER stress through metabolic impairment with AMPK activation, which in turn increases Aβ levels by a JNK3-mediated mechanism, leading to cognitive alterations. Aβ might directly potentiate the initial mitochondrial dysfunction/metabolic impairment that can be further enhanced by the deadly ER-mitochondria calcium transfer that we demonstrated to occur during ER stress conditions triggered by Aβ (Ferreiro et al., 2008; Costa et al., 2010).

    Finally, the authors identify the ER stress-activated JNK3 as a promising new target of therapeutic intervention in AD. Targeting brain UPR could be a potential therapeutic avenue for the treatment of AD; however, the functional implications of UPR activation and the clinical outcome are presently unknown. Activation of the adaptive branches of the UPR can protect neurons by increasing protein folding, protein quality control, and autophagy, but, on the other hand, UPR activation may represent a signal for neurodegeneration triggered by chronic ER stress, resulting in irreversible neuronal damage and apoptosis. Therefore, the long-term consequences of targeting UPR for reducing neuronal loss must be properly addressed. Targeting JNK3, as proposed, might avoid these potentially deleterious side effects, since its activation occurs downstream of UPR activation.

    By gaining a better understanding of the mechanisms underlying UPR activation and how it affects the neurodegenerative process in this disease, we should gain new insights into the pathological mechanisms, thus providing interesting and novel targets for disease intervention.

    References:

    Resende R, Ferreiro E, Pereira C, Resende de Oliveira C. Neurotoxic effect of oligomeric and fibrillar species of amyloid-beta peptide 1-42: involvement of endoplasmic reticulum calcium release in oligomer-induced cell death. Neuroscience. 2008 Aug 26;155(3):725-37. PubMed.

    Costa RO, Ferreiro E, Cardoso SM, Oliveira CR, Pereira CM. ER stress-mediated apoptotic pathway induced by Abeta peptide requires the presence of functional mitochondria. J Alzheimers Dis. 2010;20(2):625-36. PubMed.

    Ferreiro E, Oliveira CR, Pereira CM. The release of calcium from the endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway. Neurobiol Dis. 2008 Jun;30(3):331-42. PubMed.

    Costa RO, Lacor PN, Ferreira IL, Resende R, Auberson YP, Klein WL, Oliveira CR, Rego AC, Pereira CM. Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N-methyl-d-aspartate receptor in mature hippocampal cultures treated with amyloid-β oligomers. Aging Cell. 2012 Oct;11(5):823-33. PubMed.

    Ferreira IL, Bajouco LM, Mota SI, Auberson YP, Oliveira CR, Rego AC. Amyloid beta peptide 1-42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures. Cell Calcium. 2012 Feb;51(2):95-106. PubMed.

    View all comments by Claudia Pereira

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References

News Citations

  1. First Responder to Axon Injury Does More Harm Than Good
  2. JNK Clogs Axonal Transport
  3. Aβ Assault on Neurons Targets ER, Calcium

Webinar Citations

  1. Clearing the Fog Around Aβ Oligomers

Paper Citations

  1. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, Ron D. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science. 2000 Jan 28;287(5453):664-6. PubMed.
  2. Hunot S, Vila M, Teismann P, Davis RJ, Hirsch EC, Przedborski S, Rakic P, Flavell RA. JNK-mediated induction of cyclooxygenase 2 is required for neurodegeneration in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):665-70. PubMed.
  3. Beffert U, Nematollah Farsian F, Masiulis I, Hammer RE, Yoon SO, Giehl KM, Herz J. ApoE receptor 2 controls neuronal survival in the adult brain. Curr Biol. 2006 Dec 19;16(24):2446-52. PubMed.
  4. Li QM, Tep C, Yune TY, Zhou XZ, Uchida T, Lu KP, Yoon SO. Opposite regulation of oligodendrocyte apoptosis by JNK3 and Pin1 after spinal cord injury. J Neurosci. 2007 Aug 1;27(31):8395-404. PubMed.
  5. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell. 2008 Apr 25;30(2):214-26. PubMed.
  6. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell. 2003 Nov 26;115(5):577-90. PubMed.
  7. Muresan Z, Muresan V. c-Jun NH2-terminal kinase-interacting protein-3 facilitates phosphorylation and controls localization of amyloid-beta precursor protein. J Neurosci. 2005 Apr 13;25(15):3741-51. PubMed.
  8. Cavalli V, Kujala P, Klumperman J, Goldstein LS. Sunday Driver links axonal transport to damage signaling. J Cell Biol. 2005 Feb 28;168(5):775-87. PubMed.

Other Citations

  1. Tg2576 mice

External Citations

  1. 5xFAD transgenic mice

Further Reading

Primary Papers

  1. Yoon SO, Park DJ, Ryu JC, Ozer HG, Tep C, Shin YJ, Lim TH, Pastorino L, Kunwar AJ, Walton JC, Nagahara AH, Lu KP, Nelson RJ, Tuszynski MH, Huang K. JNK3 perpetuates metabolic stress induced by Aβ peptides. Neuron. 2012 Sep 6;75(5):824-37. PubMed.

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