22 February 2013. 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.
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. 4 Apr 2013;12:1-10. Abstract