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Abstract
Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer's disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.
Highlights
embryoid body (EB) were plated on a matrigel substrate leading to the formation of paired box protein 6 (Pax6)-expressing neuroepithelial rosettes (Supplementary Figure S2) that further differentiated to heterogeneous cultures containing non-neuronal cells (Figures 1a and b)
To quantify immunopanning efficiency, dissociated cells without immunopanning, dissociated cells isolated by neural cell adhesion molecule (NCAM) immunopanning, and dissociated cells non-adherent to the panning plates, respectively (Figure 1c), were immunocytochemically stained for NCAM and the neuronal marker microtubule-associated protein 2 (MAP2) 1 day after immunopurification (Figures 1d and e)
The fraction of MAP2-positive cells was strongly increased in cells isolated by NCAM immunopanning (91.2 ± 4.3%) as compared with control cells (28.1 ± 20.6%) and to cells nonadherent to the panning plates (12.2 ± 7.4%) (Figure 1g)
Summary
Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer's disease (AD). To investigate whether functional synapses in iPSC-derived human neurons are susceptible to the deleterious effects of Aβ, we added Aβ contained in the supernatant from cultures of 7PA2 Chinese hamster ovary (CHO) cells (expressing human APP751 carrying the familial amyloid precursor protein (APP) V717F mutation)[5,35,36] to hiPSC-derived neurons at 8 weeks after immunopurification.
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