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Modelling AD in iPSCs Provides Therapeutic Clues

“Modeling Alzheimer’s Disease with iPSCs Reveals Stress Phenotypes Associated with Intracellular Aß and Differential Drug Responsiveness”

Oligomerisation of amyloid-β peptide (Aβ) leading to amyloid plaques in the brain is thought to play a role in the pathogenesis of Alzheimer’s disease (AD) in humans (Kuo et al,  Noguchi et al and Shankar et al) but the mechanism involved is still unclear. Induced pluripotent stem cell (iPSC) technology now provides a means to study the development of this disease and the effects of Aβ oligomers and will also allow for the screening of therapeutic drugs. To this end, researchers from the laboratory of Nobuhisa Iwata and Haruhisa Inoue have reported the derivation and neuronal/astroglial differentiation of iPSCs derived from patients carrying various AD-associated genetic mutations, and have found that Aβ oligomers are not proteolytically resistant and that docosahexaenoic acid (DHA) treatment attenuated cellular stress phenotypes of AD neural cells containing Aβ oligomers (Kondo et al).

iPSCs were generated using episomal vectors from control samples, 5 familiar AD samples with amyloid beta precursor protein (APP) mutations (APP-E693D and APP-V717L) and two sporadic AD samples. These were then differentiated towards cortical neurons by modification of a previously established protocol (Morizane et al). Resulting neurons from all samples expressed cortical neuron subtype markers (SATB2 and TBR1) and were deemed functionally active. Analysis of the extra- and intracellular Aβ forms found decreased levels of total Aβ in the APP-E693D mutated samples, but increased extracellular Aβ levels in the APP-V717L samples suggesting an effect of mutation site on APP metabolism in AD. The sporadic AD samples had decreased intra-cellular Aβ levels and no change in extracellular levels. Levels of α- and β-secretase-mediated APP processing remained unaltered in all neural cells and soluble APPβ production was strongly inhibited by treatment with β-secretase inhibitor IV (BSI). The Aβ-oligomer-specific antibody NU1 was used to demonstrate that Aβ oligomers were present in the neurons and astrocytes of APP-E693Δ samples, and in one of the sporadic AD cases, at levels higher than in control cells but were absent in the fibroblasts used to generate the iPSCs. Further analysis demonstrated that Aβ oligomer-positive areas in the neurons were positive for an endoplasmic reticulum marker (BiP), an early endosomal marker (EEA1) and a lysosomal marker (LAMP2). This accumulation was inhibited by BSI treatment, with levels of Aβ-oligomers returning to control levels after 8 hours.

Gene expression profiles of control and AD neural cells found that oxidative-stress-related categories were upregulated in AD neural cells, whereas glycosylation-related categories were downregulated suggesting a perturbation in endoplasmic reticulum and Golgi function in AD neural cells and the provocation of antioxidant stress response by Aβ oligomer formation (in the APP-E693Δ and one sporadic sample), which was further confirmed through the visualization of increased reactive oxygen species (ROS) in these samples. Drugs known to improve endoplasmic reticulum stress or to inhibit ROS generation (DHA, dibenzoylmethane and NSC23766) were then tested in these cells. Only DHA treatment demonstrated positive results by reversing the phenotype observed for the APP-E693Δ and sporadic AD neural cells and leading to increased survival of these cells.

Overall, this study proves the existence of intracellular Aβ oligomers in neural cells arising from AD patient-derived iPSCs which can be used for disease modelling and drug testing.   This study suggests that Aβ oligomers contribute to neural cell death by increasing cellular stress and that the reversal of this stress through DHA treatment is a possible therapeutic avenue for AD treatment. Furthermore, Aβ oligomers were also shown to be degraded with relative ease following treatment with β-secretase inhibitor IV, a surprising finding as Aβ oligomers had been assumed to be proteolytically resistant. Lastly, the authors note that the formation of Aβ oligomers and their cellular/extracellular appearance seem to be somewhat heterogeneous between different AD iPSC lines generated, suggesting that patient-specific iPSCs would provide specific disease pathogenesis and could allow the better evaluation of drug and patient classification of AD.

 

References

  • Kuo, Y.M. et al. (1996). Water-soluble Abeta (N-40, N-42) oligomers in normal and Alzheimer disease brains. J. Biol. Chem. 271, 4077–4081.
  • Morizane, A. et al. (2011). Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells. J. Neurosci. Res. 89, 117–126.
  • Noguchi, A. et al. (2009). Isolation and characterization of patient-derived, toxic, high mass amyloid b-protein (Abeta) assembly from Alzheimer disease brains. J. Biol. Chem. 284, 32895–32905.
  • Shankar, G.M. et al. (2008). Amyloid-b protein dimers isolated directly from Alzheimer’s brains impair synaptic plasticity and memory. Nat. Med. 14, 837–842.

Study originally appeared in Cell Stem Cell.

Stem Cell Correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.