Ironing Out Apoptotic Role for New APP-Binding Protein

In a hunt for molecules that bind amyloid-β precursor protein (APP), researchers have discovered a mitochondrial ion transporter that promotes programmed cell death in neurons. Dubbed “appoptosin,” the protein drives synthesis of heme, the iron-containing porphyrin in hemoglobins. The study found unusually high levels of appoptosin in the brains of people with stroke or Alzheimer’s disease, and blocking its expression in cultured cells prevented Aβ-induced morphological changes and cell death. The authors, led by Huaxi Xu, Sanford-Burnam Medical Research Institute, La Jolla, California, propose that interaction of appoptosin with APP suppresses cell death by limiting mitochondrial heme, which can generate reactive oxygen species and potentially cause oxidative stress, inflammation, and other cellular damage. The research appeared in the October 31 Journal of Neuroscience.

Xu and colleagues’ search for APP partners broadened when they identified a tumor suppressor role for the intracellular chunk of APP produced by γ-secretase cleavage. The APP intracellular domain (AICD) bound the promoter of epidermal growth factor receptor (EGFR), curbing its transcription (Zhang et al., 2007). EGFR is upregulated in various cancers, and Xu said the AICD data seem to fit with the discovery of increased skin cancer rates among treated participants in the halted Phase 3 AD trial of Eli Lilly’s γ-secretase inhibitor (see ARF related news story). Xu’s team did a yeast two-hybrid screen for AICD-associated proteins—one of which turned out to be appoptosin. Xu previously reported on this protein, then called 168, at the 2007 Society for Neuroscience meeting in San Diego (see ARF conference story), and has been characterizing the protein ever since.

At the time, Han Zhang, Xiamen University, Fujian, China, first author on the paper, and colleagues could not find any published data on this protein. But they found that it promoted apoptosis: Overexpressing the gene triggered death in human embryonic kidney cells, and knocking it out made neurons more resistant to Aβ toxicity. Further experiments honed the researchers’ focus by showing that the AICD-binding protein induced cell death through mitochondrial caspase-3. Still, Xu said, they wondered how this protein causes apoptosis.

A 2009 paper provided the critical hint. Other scientists linked a rare form of anemia to mutations in a mitochondrial gene (SLC25A38) that overlapped with the p168 sequence from Xu’s yeast two-hybrid screen (Guernsey et al., 2009). Xu then suspected the mutated gene might be important for producing heme.

Consistent with Xu’s hunch, heme levels rose, as did amounts of cleaved caspase-3, in mouse neuroblastoma cells transfected with appoptosin. Treatment with a heme synthesis inhibitor prevented the caspase-3 activation. Furthermore, overexpression of appoptosin caused human neuroblastoma cells to overproduce reactive oxygen species (ROS), and weakened their mitochondrial membrane potential. Addition of Aβ1-42 and glutamate ramped up heme and cleaved caspase-3 levels—but not if appoptosin was downregulated by RNA interference (RNAi). Plus, appoptosin knockdown prevented neuritic damage, mitochondrial fragmentation, and other morphological abnormalities typically induced by Aβ treatment. The findings show that appoptosin promotes cell death by turning up heme synthesis, whereas downregulating appoptosin helps cells resist neurotoxicity.

How does APP fit in? Previous work suggested a connection between AICD and caspase activation (see Park et al., 2009; ARF conference story; Kajiwara et al., 2009). In the present study, Zhang and colleagues looked at caspase-3 activation in HEK293T human embryonic kidney cells transfected with appoptosin and different forms of APP—full length or one of several truncation mutants. Overexpression of APP relieved appoptosin-induced caspase-3 cleavage in all cases but one—the APP mutant lacking the 57-amino-acid intracellular domain that binds appoptosin. Curiously, expression of this intracellular fragment (APP C57)—the only non-membrane-tethered form of APP used in the study—also had little influence on caspase-3 activation. These findings suggested to the authors that membrane-anchored APP binds to appoptosin and keeps it in the cytosol, reducing its transport into mitochondria where it can cause heme overproduction and apoptosis. But “when membrane-anchored APP is cleaved by γ-secretase, APP-bound appoptosin is released together with APP C57/AICD and translocated to the mitochondria to exert its function,” the authors write. In support of this idea, fluorescence microscopy of HEK293T transfectants showed that appoptosin binds full-length APP primarily in the cytosol, whereas association between appoptosin and APP C57 mainly occurs in mitochondria.

“This is an important study,” said Ashley Bush of the Mental Health Research Institute, Victoria, Australia. The findings seem to jibe with recent work suggesting that neurons rack up hemoglobin in AD in response to hypoxia (Chuang et al., 2012), Bush noted in an e-mail to Alzforum. From immunoblot analyses in the present work, the authors detected more appoptosin and cleaved caspase-3 in cortical extracts from 10 AD patients and four stroke patients, relative to brain samples of cognitively healthy controls. There are other potential links to dementia. Heme metabolism falters in AD and other neurodegenerative disorders (Ryter and Tyrrell, 2000; Atamna, 2004), and iron chelators are emerging as potential therapeutic approaches because they can prevent iron-based reactions that lead to reactive oxygen species (see ARF conference story). The new data also appear consistent with research by Bush and colleagues highlighting iron transport roles for APP and tau (ARF related news story on Duce et al., 2010; ARF related news story on Lei et al., 2012). Moreover, appoptosin expression correlates with polymorphisms in or near MOBP (myelin-associated oligodendrocyte basic protein)—a new gene linked to risk for the tauopathy progressive supranuclear palsy (Höglinger et al., 2011).

So far, apoptosis genes have not been tied to increased AD risk, making it hard to connect the disease with programmed cell death. The current work “provides what is perhaps the most direct molecular pathway linking APP metabolism and apoptosis,” noted Sam Gandy of Mount Sinai School of Medicine, New York, in an e-mail to Alzforum (see full comment below). The data suggest that reducing brain appoptosin could be a good therapeutic strategy for AD, he wrote.

To get a better handle on mechanisms underlying appoptosin upregulation during disease or injury, Xu’s team has made appoptosin knockout mice and are crossing appoptosin-overexpressing mice with mice that model AD or other neurodegenerative disorders. They are also trying to develop compounds that inhibit appoptosin transcription or increase its interaction with APP.—Esther Landhuis

Comments

    • User Profile Image Sam Gandy
      Icahn School of Medicine at Mount Sinai
    • Posted:

    There has been a longstanding curiosity in the field of Alzheimer's research regarding possible relationships between APP/Aβ and the process of apoptosis. Aβ oligomer toxicity is closely associated with neurotoxicity, but typical programmed cell death (aka apoptosis) has not been robustly indicated. Caspase cleavage of APP and PS2 have long stood as possible nexuses whereby APP metabolism and apoptosis might converge. Now Xu and colleagues discover a new protein, dubbed appoptosin, that bridges the gap between APP and mitochondrial physiology and apoptosis. Appoptosin levels are increased in AD brain and infarcted brain, and levels of any protein that buck the trend and rise during neuronal death are usually worth noting. Downregulation of appoptosin protects neurons from Aβ toxicity and glutamate toxicity, raising the possibility that therapeutic reduction of brain appoptosin becomes the latest novel strategy for protecting the brain besieged by AD.

    View all comments by Sam Gandy
    • User Profile Image Jack T. Rogers
      Massachusetts General Hospital/Harvard University
    • Posted:

    Zhang et al. have identified further links between Alzheimer's disease and iron metabolism via their discovery of a role for appoptosin, which they reported to be a novel amyloid precursor protein (APP)-binding protein after yeast hybrid analysis.

    Han Zhang's team collaborated with Huaxi Xu's team to conclusively show that appoptosin expression causes mitochondrial-driven apoptosis. However, more significantly, it can bind the C-terminal of the APP, tethered to the membrane. After damage, or even secretase cleavage, appoptosin moves to the mitochondria and is proposed to have a significant role in mitochondrial heme biosynthesis. Excess heme is known to generate reactive oxygen species by Fenton chemistry and thus cause neuronal death, as after hemorrhage, for example.

    Intriguing links to iron metabolism yet again arise from this tour de force since the findings are consistent with the 2010 demonstration that APP also binds ferroportin and is considered an iron export ferroxidase via its N-terminus (see Duce et al., 2010).

    Clearly, the APP/appoptosin partnership has a significant role in iron homeostasis.

    This is particularly evident by the RNA binding protein iron-regulatory protein 1 (IRP1), which controls the rate of iron/heme-dependent translation of APP to thereby efflux excess iron from neural cells at risk from heme or iron overload (Cho et al., 2010).

    The link between APP and heme/iron metabolism, and now apoptosis, is supported by more evidence that, like APP, appoptosin has a central role in iron homeostasis, and that mistakes in this homeostasis can kill neurons.

    Indeed, genetic mutations to the appoptosin gene cause recessive congenital sideroblastic anemia (Guernsey et al., 2009, a reference in this paper).

    References:

    Duce JA, Tsatsanis A, Cater MA, James SA, Robb E, Wikhe K, Leong SL, Perez K, Johanssen T, Greenough MA, Cho HH, Galatis D, Moir RD, Masters CL, McLean C, Tanzi RE, Cappai R, Barnham KJ, Ciccotosto GD, Rogers JT, Bush AI. Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell. 2010 Sep 17;142(6):857-67. PubMed.

    Cho HH, Cahill CM, Vanderburg CR, Scherzer CR, Wang B, Huang X, Rogers JT. Selective translational control of the Alzheimer amyloid precursor protein transcript by iron regulatory protein-1. J Biol Chem. 2010 Oct 8;285(41):31217-32. PubMed.

    Guernsey DL, Jiang H, Campagna DR, Evans SC, Ferguson M, Kellogg MD, Lachance M, Matsuoka M, Nightingale M, Rideout A, Saint-Amant L, Schmidt PJ, Orr A, Bottomley SS, Fleming MD, Ludman M, Dyack S, Fernandez CV, Samuels ME. Mutations in mitochondrial carrier family gene SLC25A38 cause nonsyndromic autosomal recessive congenital sideroblastic anemia. Nat Genet. 2009 Jun;41(6):651-3. PubMed.

    View all comments by Jack T. Rogers

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References

News Citations

  1. Lilly Halts IDENTITY Trials as Patients Worsen on Secretase Inhibitor
  2. San Diego: Exploring the Role of APP in Transit
  3. Chicago: APP Roles in Caspase Gene Regulation, Muscle Function?
  4. Stockholm: Therapeutics Roundup—Some New, Some Not So Much
  5. Iron Export? New Role Links APP, Metals, to Oxidative Stress
  6. Tau Joins APP in the Ironworks

Paper Citations

  1. Zhang YW, Wang R, Liu Q, Zhang H, Liao FF, Xu H. Presenilin/gamma-secretase-dependent processing of beta-amyloid precursor protein regulates EGF receptor expression. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10613-8. PubMed.
  2. Guernsey DL, Jiang H, Campagna DR, Evans SC, Ferguson M, Kellogg MD, Lachance M, Matsuoka M, Nightingale M, Rideout A, Saint-Amant L, Schmidt PJ, Orr A, Bottomley SS, Fleming MD, Ludman M, Dyack S, Fernandez CV, Samuels ME. Mutations in mitochondrial carrier family gene SLC25A38 cause nonsyndromic autosomal recessive congenital sideroblastic anemia. Nat Genet. 2009 Jun;41(6):651-3. PubMed.
  3. Park SA, Shaked GM, Bredesen DE, Koo EH. Mechanism of cytotoxicity mediated by the C31 fragment of the amyloid precursor protein. Biochem Biophys Res Commun. 2009 Oct 16;388(2):450-5. PubMed.
  4. Kajiwara Y, Akram A, Katsel P, Haroutunian V, Schmeidler J, Beecham G, Haines JL, Pericak-Vance MA, Buxbaum JD. FE65 binds Teashirt, inhibiting expression of the primate-specific caspase-4. PLoS One. 2009;4(4):e5071. PubMed.
  5. Chuang JY, Lee CW, Shih YH, Yang T, Yu L, Kuo YM. Interactions between amyloid-β and hemoglobin: implications for amyloid plaque formation in Alzheimer's disease. PLoS One. 2012;7(3):e33120. PubMed.
  6. Ryter SW, Tyrrell RM. The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and antioxidant properties. Free Radic Biol Med. 2000 Jan 15;28(2):289-309. PubMed.
  7. Atamna H. Heme, iron, and the mitochondrial decay of ageing. Ageing Res Rev. 2004 Jul;3(3):303-18. PubMed.
  8. Duce JA, Tsatsanis A, Cater MA, James SA, Robb E, Wikhe K, Leong SL, Perez K, Johanssen T, Greenough MA, Cho HH, Galatis D, Moir RD, Masters CL, McLean C, Tanzi RE, Cappai R, Barnham KJ, Ciccotosto GD, Rogers JT, Bush AI. Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell. 2010 Sep 17;142(6):857-67. PubMed.
  9. Lei P, Ayton S, Finkelstein DI, Spoerri L, Ciccotosto GD, Wright DK, Wong BX, Adlard PA, Cherny RA, Lam LQ, Roberts BR, Volitakis I, Egan GF, McLean CA, Cappai R, Duce JA, Bush AI. Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export. Nat Med. 2012 Feb;18(2):291-5. PubMed.
  10. Höglinger GU, Melhem NM, Dickson DW, Sleiman PM, Wang LS, Klei L, Rademakers R, de Silva R, Litvan I, Riley DE, van Swieten JC, Heutink P, Wszolek ZK, Uitti RJ, Vandrovcova J, Hurtig HI, Gross RG, Maetzler W, Goldwurm S, Tolosa E, Borroni B, Pastor P, , Cantwell LB, Han MR, Dillman A, van der Brug MP, Gibbs JR, Cookson MR, Hernandez DG, Singleton AB, Farrer MJ, Yu CE, Golbe LI, Revesz T, Hardy J, Lees AJ, Devlin B, Hakonarson H, Müller U, Schellenberg GD. Identification of common variants influencing risk of the tauopathy progressive supranuclear palsy. Nat Genet. 2011 Jul;43(7):699-705. PubMed.

Further Reading

Papers

  1. Duce JA, Tsatsanis A, Cater MA, James SA, Robb E, Wikhe K, Leong SL, Perez K, Johanssen T, Greenough MA, Cho HH, Galatis D, Moir RD, Masters CL, McLean C, Tanzi RE, Cappai R, Barnham KJ, Ciccotosto GD, Rogers JT, Bush AI. Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell. 2010 Sep 17;142(6):857-67. PubMed.
  2. Horowitz MP, Greenamyre JT. Mitochondrial iron metabolism and its role in neurodegeneration. J Alzheimers Dis. 2010;20 Suppl 2:S551-68. PubMed.
  3. Lei P, Ayton S, Finkelstein DI, Spoerri L, Ciccotosto GD, Wright DK, Wong BX, Adlard PA, Cherny RA, Lam LQ, Roberts BR, Volitakis I, Egan GF, McLean CA, Cappai R, Duce JA, Bush AI. Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export. Nat Med. 2012 Feb;18(2):291-5. PubMed.
  4. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012 May 25;149(5):1060-72. PubMed.

Primary Papers

  1. Zhang H, Zhang YW, Chen Y, Huang X, Zhou F, Wang W, Xian B, Zhang X, Masliah E, Chen Q, Han JD, Bu G, Reed JC, Liao FF, Chen YG, Xu H. Appoptosin is a novel pro-apoptotic protein and mediates cell death in neurodegeneration. J Neurosci. 2012 Oct 31;32(44):15565-76. PubMed.

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