Aβ Oligomers Linked to ER Stress in Patient-Derived Neurons

Research published in the February 21 Cell Stem Cell solidifies the case that human neurons made from reprogrammed patient cells can help scientists unravel pathological mechanisms behind Alzheimer's disease and evaluate potential therapies. In previous work with neurons generated from induced pluripotent stem cells (iPSCs), researchers found more Aβ secreted from AD patient-derived neurons relative to age-matched control cells. The current study focuses on intracellular Aβ. The authors report that neurons made from familial and sporadic AD iPSC lines racked up Aβ oligomers, which triggered endoplasmic reticulum (ER) and oxidative stress. In some of those cells, docosahexaenoic acid provided relief. Haruhisa Inoue of Kyoto University and Nobuhisa Iwata of Nagasaki University, both in Japan, led the research.

Although the technology for transforming adult cells into a pluripotent state made its debut back in 2006, it was only two years ago that scientists reported using iPSC-derived neurons to study Alzheimer’s disease mechanisms. In AD iPSC studies to date, derived neurons churned out more Aβ than those differentiated from iPSCs from healthy controls. Scientists saw such effects in a range of cell models, including several expressing familial AD mutations in presenilin (ARF related news story on Qiang et al., 2011; Yagi et al., 2011) or amyloid precursor protein (APP) (ARF related news story on Israel et al., 2012), and cells from Down's syndrome patients, who have an extra copy of APP (Shi et al., 2012). A similar phenotype showed up in neurons derived from iPSCs of a sporadic AD patient (Israel et al., 2012), suggesting altered APP processing as a common mechanism for sporadic and familial forms of AD.

In the current paper, first author Takayuki Kondo and colleagues characterized neurons derived from two people with sporadic AD and two with familial AD APP mutations—V717L and E693Δ. The latter is unusual. Deletion of this glutamate promotes Aβ oligomerization; however, people with the APP-E693Δ mutation show no extracellular Aβ deposits on amyloid PET imaging, even while developing symptoms of early onset AD (ARF related conference story on Tomiyama et al., 2008; Shimada et al., 2011). In like fashion, transgenic mice overexpressing APP-E693Δ accumulate intracellular Aβ oligomers but no plaques (ARF related news story on Tomiyama et al., 2010).

Using the NU1 anti-Aβ oligomer antibody, the researchers report that oligomers accumulated in neurons derived from APP-E693Δ iPSCs, and from one of the sporadic AD iPSC lines. The scientists saw no oligomers in V717L FAD neurons or in the second sporadic AD line. Some scientists said that intracellular oligomers are hard to detect and quantify, and noted that the NU1 antibody may lack specificity for Aβ oligomers. Nonetheless, the work “is interesting because it provides more evidence for aggregation inside the cell as being related to pathogenesis,” Charles Glabe of the University of California, Irvine, wrote in an e-mail to Alzforum.

Oligomers resided in the ER and in endosomal and lysosomal compartments. This was based on immunostaining with organelle-specific antibodies, and hints that the Aβ aggregates might perturb ER and Golgi function. In support of that idea, microarray analyses showed that peroxidase activity and genes related to oxidative stress were upregulated. Gene expression related to glycosylation—a post-translational modification that occurs in the ER/Golgi—fell in APP-E693Δ neurons relative to control cells.

To test if Aβ oligomers might bring about these changes, the researchers treated the cells with a β-secretase inhibitor. This not only did away with the oligomers, but it also corrected ER and Golgi protein expression—decreasing levels of BiP and cleaved caspase-4, and counteracting increases in peroxiredoxin-4 and reactive oxygen species. These data indicate “that intracellular Aβ oligomers provoke ER and oxidative stress, the increase in reactive oxygen species (ROS) most likely occurring via a vicious cycle between ER and oxidative stress,” the authors write.

Interestingly, docosahexaenoic acid (DHA), an omega-3 fatty acid and antioxidant, also normalized ER and Golgi protein expression. Furthermore, DHA reduced production of ROS and improved cell survival, without changing Aβ oligomer or extracellular Aβ levels. The two neuron lines lacking Aβ oligomers failed to respond to DHA treatment. This fish oil has generated interest as a potential therapy. While epidemiological evidence links a DHA-rich diet with reduced AD risk (see ARF related news story), the fatty acid failed to curb cognitive decline in an 18-month trial of 402 AD patients (ARF related news story on Quinn et al., 2010).

Selina Wray of University College London, U.K., found the differential responsiveness to DHA the most interesting aspect of this paper. She said that understanding why only certain cell lines respond to certain treatments could have implications in the clinic. “The success of a particular treatment could depend on patients being ‘subtyped’ appropriately,” she noted in an e-mail to Alzforum (see full comment below). The authors suggest their data could explain why DHA treatment might only be effective in some AD patients, namely, those with intracellular Aβ oligomers. Greg Cole of the University of California, Los Angeles, noted that the study used realistic DHA doses, and protection shown by the lowest dose (1 microM) was physiologically relevant.

On a broader level, Asa Abeliovich of Columbia University, New York—whose lab first published on iPSC-derived neurons from familial AD patients—finds it noteworthy that several groups have now reported changes in APP processing in human AD cell models. “It is important to look at a variety of mutations,” Abeliovich said. “It is exciting that [the current study] looked at a clinically intriguing mutation (i.e., APP-E693Δ).”

While iPSC technology continues to develop (see ARF related series), the original enabling discovery netted $3 million for Shinya Yamanaka of Kyoto University, Japan, and the Gladstone Institutes in San Francisco. Yamanaka was among 11 scientists named winners of the inaugural Breakthrough Prize in Life Sciences (see Science article).—Esther Landhuis

Comments

    • User Profile Image Selina Wray
      Institute of Neurology, University College London
    • Posted:

    This paper adds to a growing number of publications using iPSCs to study AD-related cellular phenotypes in vitro (Qiang et al., 2011; Israel et al., 2012; Yagi et al., 2011; Shi et al., 2012). It is reassuring to see so many labs independently finding robust phenotypes in their various cell lines. The fact that these cells converge on similar phenotypes with respect to altered APP processing is interesting, and I think we can now be confident that patient-derived neurons are a good model for AD pathogenesis. The next step is to determine the mechanism(s) by which these observed differences in APP processing are leading to cell death in AD. I hope we'll start to see reports where patient-derived neurons are being used to uncover novel disease mechanisms.

    For me, the most interesting aspect of this paper is the differential responsiveness to DHA. Understanding why certain cell lines are responsive to treatments whilst others are not could ultimately have implications in the clinic: The success of a particular treatment could depend on patients being "subtyped" appropriately. However, given that this study only examines cells from two familial patients and two sporadic patients, it is difficult to draw any firm conclusions in that respect without expanding this study to include more patients.

    References:

    Qiang L, Fujita R, Yamashita T, Angulo S, Rhinn H, Rhee D, Doege C, Chau L, Aubry L, Vanti WB, Moreno H, Abeliovich A. Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons. Cell. 2011 Aug 5;146(3):359-71. PubMed. RETRACTED

    Israel MA, Yuan SH, Bardy C, Reyna SM, Mu Y, Herrera C, Hefferan MP, Van Gorp S, Nazor KL, Boscolo FS, Carson CT, Laurent LC, Marsala M, Gage FH, Remes AM, Koo EH, Goldstein LS. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature. 2012 Jan 25;482(7384):216-20. PubMed.

    Yagi T, Ito D, Okada Y, Akamatsu W, Nihei Y, Yoshizaki T, Yamanaka S, Okano H, Suzuki N. Modeling familial Alzheimer's disease with induced pluripotent stem cells. Hum Mol Genet. 2011 Dec 1;20(23):4530-9. PubMed.

    Shi Y, Kirwan P, Smith J, Maclean G, Orkin SH, Livesey FJ. A human stem cell model of early Alzheimer's disease pathology in Down syndrome. Sci Transl Med. 2012 Mar 7;4(124):124ra29. PubMed.

    View all comments by Selina Wray

  2. This extensive, collaborative study brings new evidence supporting the idea that the pathologically relevant amyloid-β peptide (Aβ) is produced and aggregates in compartments in the neuronal soma, including the endosomes, lysosomes, and the endoplasmic reticulum (ER). The generation and oligomerization of Aβ in the ER does not come as a surprise, given that the ER is one of the centers of stress response of the cell. Many types of stress—oxidative, improper protein folding, or the accumulation of proteins in the soma due to impeded transport along the secretory pathway—appear to be sensed by the ER. The ER response, which is geared to relieve the stress-related pathology, is currently widely studied.

    The idea that Aβ is generated in the soma, where it accumulates and oligomerizes, is not new (1-3). Also, the idea that stress leads to the generation, accumulation, and oligomerization of Aβ in the endoplasmic reticulum has been previously proposed. For example, we reported that enhanced cleavage of APP in the ER, followed by accumulation and oligomerization of Aβ inside the ER, represents the specific response of the neuron to impeded axonal transport (4)—a situation that becomes an issue in old age (5). It is also likely that part of the oligomeric Aβ produced either in the ER or in the somatic endosomes escapes from these compartments and is transported into neurites, accumulating at their distal tips (2). Although some Aβ could certainly be generated and aggregated at the synapse, emerging evidence, such as that provided by this study, highlights the possibility that pathologically relevant Aβ oligomers are produced in the neuronal soma in neurons affected by Alzheimer's disease. This intraneuronal Aβ could be released in the extracellular space either by cell death or by other mechanisms operating in neurons, such as externalization via exosomes. The field of Alzheimer’s disease is eagerly awaiting new developments in this direction.

    References:

    Cook DG, Forman MS, Sung JC, Leight S, Kolson DL, Iwatsubo T, Lee VM, Doms RW. Alzheimer's A beta(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat Med. 1997 Sep;3(9):1021-3. PubMed.

    Muresan Z, Muresan V. Neuritic deposits of amyloid-beta peptide in a subpopulation of central nervous system-derived neuronal cells. Mol Cell Biol. 2006 Jul;26(13):4982-97. PubMed.

    Skovronsky DM, Doms RW, Lee VM. Detection of a novel intraneuronal pool of insoluble amyloid beta protein that accumulates with time in culture. J Cell Biol. 1998 May 18;141(4):1031-9. 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.

    Muresan V, Muresan Z. Is abnormal axonal transport a cause, a contributing factor or a consequence of the neuronal pathology in Alzheimer's disease?. Future Neurol. 2009 Nov 1;4(6):761-773. PubMed.

    View all comments by Virgil Muresan

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References

News Citations

  1. Alzheimer’s Neurons Made to Order: Direct Conversion From Skin Cells
  2. Induced Neurons From AD Patients Hint at Disease Mechanisms
  3. San Diego: Oligomers Live Up to Bad Reputation, Part 2
  4. New APP Model: No Plaques—Plenty of Pathology
  5. Talking Fish Fat And Cholesterol
  6. Paper Alert: Negative DHA Trial Fuels Soul-Searching in AD Field
  7. Where in the World Are the iPS Cells?

Paper Citations

  1. Qiang L, Fujita R, Yamashita T, Angulo S, Rhinn H, Rhee D, Doege C, Chau L, Aubry L, Vanti WB, Moreno H, Abeliovich A. Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons. Cell. 2011 Aug 5;146(3):359-71. PubMed. RETRACTED
  2. Yagi T, Ito D, Okada Y, Akamatsu W, Nihei Y, Yoshizaki T, Yamanaka S, Okano H, Suzuki N. Modeling familial Alzheimer's disease with induced pluripotent stem cells. Hum Mol Genet. 2011 Dec 1;20(23):4530-9. PubMed.
  3. Israel MA, Yuan SH, Bardy C, Reyna SM, Mu Y, Herrera C, Hefferan MP, Van Gorp S, Nazor KL, Boscolo FS, Carson CT, Laurent LC, Marsala M, Gage FH, Remes AM, Koo EH, Goldstein LS. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature. 2012 Jan 25;482(7384):216-20. PubMed.
  4. Shi Y, Kirwan P, Smith J, Maclean G, Orkin SH, Livesey FJ. A human stem cell model of early Alzheimer's disease pathology in Down syndrome. Sci Transl Med. 2012 Mar 7;4(124):124ra29. PubMed.
  5. Tomiyama T, Nagata T, Shimada H, Teraoka R, Fukushima A, Kanemitsu H, Takuma H, Kuwano R, Imagawa M, Ataka S, Wada Y, Yoshioka E, Nishizaki T, Watanabe Y, Mori H. A new amyloid beta variant favoring oligomerization in Alzheimer's-type dementia. Ann Neurol. 2008 Mar;63(3):377-87. PubMed.
  6. Shimada H, Ataka S, Tomiyama T, Takechi H, Mori H, Miki T. Clinical course of patients with familial early-onset Alzheimer's disease potentially lacking senile plaques bearing the E693Δ mutation in amyloid precursor protein. Dement Geriatr Cogn Disord. 2011;32(1):45-54. PubMed.
  7. Tomiyama T, Matsuyama S, Iso H, Umeda T, Takuma H, Ohnishi K, Ishibashi K, Teraoka R, Sakama N, Yamashita T, Nishitsuji K, Ito K, Shimada H, Lambert MP, Klein WL, Mori H. A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo. J Neurosci. 2010 Apr 7;30(14):4845-56. PubMed.
  8. Quinn JF, Raman R, Thomas RG, Yurko-Mauro K, Nelson EB, Van Dyck C, Galvin JE, Emond J, Jack CR, Weiner M, Shinto L, Aisen PS. Docosahexaenoic acid supplementation and cognitive decline in Alzheimer disease: a randomized trial. JAMA. 2010 Nov 3;304(17):1903-11. PubMed.

External Citations

  1. Science article

Further Reading

Papers

  1. Qiang L, Fujita R, Yamashita T, Angulo S, Rhinn H, Rhee D, Doege C, Chau L, Aubry L, Vanti WB, Moreno H, Abeliovich A. Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons. Cell. 2011 Aug 5;146(3):359-71. PubMed. RETRACTED
  2. Yagi T, Ito D, Okada Y, Akamatsu W, Nihei Y, Yoshizaki T, Yamanaka S, Okano H, Suzuki N. Modeling familial Alzheimer's disease with induced pluripotent stem cells. Hum Mol Genet. 2011 Dec 1;20(23):4530-9. PubMed.
  3. Israel MA, Yuan SH, Bardy C, Reyna SM, Mu Y, Herrera C, Hefferan MP, Van Gorp S, Nazor KL, Boscolo FS, Carson CT, Laurent LC, Marsala M, Gage FH, Remes AM, Koo EH, Goldstein LS. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature. 2012 Jan 25;482(7384):216-20. PubMed.
  4. Shi Y, Kirwan P, Smith J, Maclean G, Orkin SH, Livesey FJ. A human stem cell model of early Alzheimer's disease pathology in Down syndrome. Sci Transl Med. 2012 Mar 7;4(124):124ra29. PubMed.
  5. Egawa N, Kitaoka S, Tsukita K, Naitoh M, Takahashi K, Yamamoto T, Adachi F, Kondo T, Okita K, Asaka I, Aoi T, Watanabe A, Yamada Y, Morizane A, Takahashi J, Ayaki T, Ito H, Yoshikawa K, Yamawaki S, Suzuki S, Watanabe D, Hioki H, Kaneko T, Makioka K, Okamoto K, Takuma H, Tamaoka A, Hasegawa K, Nonaka T, Hasegawa M, Kawata A, Yoshida M, Nakahata T, Takahashi R, Marchetto MC, Gage FH, Yamanaka S, Inoue H. Drug screening for ALS using patient-specific induced pluripotent stem cells. Sci Transl Med. 2012 Aug 1;4(145):145ra104. PubMed.

Primary Papers

  1. Kondo T, Asai M, Tsukita K, Kutoku Y, Ohsawa Y, Sunada Y, Imamura K, Egawa N, Yahata N, Okita K, Takahashi K, Asaka I, Aoi T, Watanabe A, Watanabe K, Kadoya C, Nakano R, Watanabe D, Maruyama K, Hori O, Hibino S, Choshi T, Nakahata T, Hioki H, Kaneko T, Naitoh M, Yoshikawa K, Yamawaki S, Suzuki S, Hata R, Ueno S, Seki T, Kobayashi K, Toda T, Murakami K, Irie K, Klein WL, Mori H, Asada T, Takahashi R, Iwata N, Yamanaka S, Inoue H. Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated with intracellular Aβ and differential drug responsiveness. Cell Stem Cell. 2013 Apr 4;12(4):487-96. Epub 2013 Feb 21 PubMed.

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