Research articleIncreased susceptibility to Aβ toxicity in neuronal cultures derived from familial Alzheimer’s disease (PSEN1-A246E) induced pluripotent stem cells
Introduction
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease leading to dementia in the elderly population. Currently, over 35 million individuals worldwide and almost 6 million Americans suffer from AD, representing the sixth-leading cause of death [10], [12]. AD is characterized by cerebral atrophy that occurs as a consequence of extensive neurodegeneration. The typical abnormalities observed in the brain of AD patients include synaptic alterations, brain inflammation and the presence of extracellular protein aggregates in the form of amyloid plaques composed predominantly of the amyloid β (Aβ) peptide and intracellular protein aggregates in the form of neurofibrillary tangles which consist primarily of an abnormally hyper-phosphorylated form of the microtubule-associated protein tau [37].
A growing body of evidence suggests that small oligomeric Aβ species are the primary toxic agents driving the synaptic abnormalities and the extensive neuronal death observed in AD. In recent years, a large effort has been focused on understanding the underlying mechanisms for the toxicity of these Aβ oligomers. Many mechanisms have been proposed including cell membrane damage, aberrant signal transduction, endoplasmic reticulum stress, disturbance of autophagy, mitochondrial dysfunction, oxidative stress, among others [18], [40]. Most of the studies have been performed using diverse transgenic mice, primary cultures of rodent and human nerve tissue, or immortalized cell lines (e.g. PC12, HEK293 and SH-SY5Y) [8], [13], [16], [29], [35], [44]. Despite considerable progress in understanding the disease mechanisms using these models, the knowledge gained has not been translated into the development of efficient therapies. For this reason, there is an urgent need to develop new models that may provide more predictable information regarding the mechanism of damage to human nerve cells.
An emerging and promising cellular approach to model human disease are the use of induced pluripotent stem cells (iPSCs). iPSCs are pluripotent cells derived from somatic cells by the ectopic expression of key reprogramming factors under embryonic stem cell (ESC) culture conditions [47], [48]. Like ESCs, iPSCs are undifferentiated cells able to self-renew in vitro and generate cells derived from the three primary germ layers of the embryo. Interestingly, differentiated cells derived from iPSCs of patients are able to recapitulate in vitro key pathological features of many diseases, including neurodegenerative disorders such as AD [14], [20], [41], [55], [59]. Particularly, neurons derived from AD iPSCs (fAD, sAD) and Down syndrome patients exhibit higher levels of cellular stress, phosphorylated tau, Aβ, as well as the presence of intra- and extracellular amyloid aggregates [15], [20], [46]. For these reasons, AD iPSC-derived neurons might be more susceptible to the exposure of Aβ oligomers compared with cells coming from healthy individuals. To test this hypothesis, we studied the toxic effects of Aβ1-42 oligomers using neuronal cultures derived from iPSCs of a healthy individual, as well as fAD and sAD patients. We found that neurons derived from fAD iPSCs have a higher susceptibility to toxicity from Aβ1-42 oligomers compared to similar neurons originated from healthy and sAD iPSCs. Our findings suggests that fAD neurons have intrinsic properties that make them more prone to be damaged in an AD brain environment, highlighting the importance of using patients’ cells in vitro to screen potential neuroprotective agents for AD.
Section snippets
Patients and fibroblast culture
Human skin fibroblasts were obtained from Coriell Cell Repositories (Coriell, Candem, NJ, USA). The donors included: 1) A 56 years old male patient with fAD (cell number: AG06840) carrying a missense mutation (A246E) in the presenilin 1 (PSEN1) gene linked to early-onset of AD [45]; 2) A 59 years old male patient with sAD (cell number: AG06844) and 3) A 66 year old female (unaffected spouse of an AD family member) whose cells were used to generate a healthy iPSC line (cell number: AG08517). Upon
Generation of iPSCs, iPSC-NPCs and neurons
To develop a human cellular model of AD in vitro, we derived iPSCs from skin fibroblasts obtained from a healthy individual, a fAD (mutated PSEN1) patient and a sAD patient. 30 days after transduction, small stem cell-like colonies were manually picked from the culture dishes and passaged for expansion as previously described [34]. The expanded cells formed flat colonies with smooth edges composed of highly packed cells with large nuclei and a small amount of cytoplasm (Fig. 1A–C). iPSC colonies
Discussion
The mechanism and species responsible for Aβ neurotoxicity remains elusive. Prior to the establishment of iPSCs, cellular models to explore the role of the Aβ peptide in the pathophysiology of AD were largely limited to: 1) mouse and human immortalized cell lines transfected to express mutant genes associated with fAD cases and 2) primary cultures (either as mono-cultures or co-cultures with microglia) of rodent and human tissue that were usually obtained at the embryonic stage [8], [13], [16],
Conclusions
Our approach with AD specific iPSCs allowed us to test the toxicity of Aβ oligomers in a human cellular model, possessing the same genetic background as the patients. Our results suggest that AD neurons, in particularly those from a fAD patient, were more sensitive to the toxic effects of Aβ oligomers than cells coming from sAD or healthy controls. Taken together, this work highlights the importance to use neurons derived from AD iPSCs in drug screening in order to develop new strategies to
Acknowledgements
We would like to thanks Dr. Charles Mays (University of Texas Medical School at Houston) for critically reviewing the manuscript. This work was funded in part by a grant from the UT Brain Initiative to CS.
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