Increased susceptibility to Aβ toxicity in neuronal cultures derived from familial Alzheimer’s disease (PSEN1-A246E) induced pluripotent stem cells

doi:10.1016/j.neulet.2016.12.060

Neuroscience Letters

Volume 639, 3 February 2017, Pages 74-81

https://doi.org/10.1016/j.neulet.2016.12.060 Get rights and content

Highlights

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Induced pluripotent stem cells (iPSCs) were produced from healthy individuals and Alzheimer’s disease patients.
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iPSCs can be successfully differentiated into neural precursors and neurons.
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iPSCs derived from familial AD patients secrete a higher concentration of Aβ1-42.
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Neurons derived from familial AD iPSCs exhibit higher susceptibility to Aβ1-42 oligomers than healthy cells.

Abstract

Alzheimer’s disease (AD) is the most common cause of late-life dementia and represents one of the leading causes of death worldwide. The generation of induced pluripotent stem cells (iPSC) has facilitated the production and differentiation of stem cells from patients somatic cells, offering new opportunities to model AD and other diseases in vitro. In this study, we generated iPSCs from skin fibroblasts obtained from a healthy individual, as well as sporadic (sAD) and familial AD (fAD, PSEN1-A246E mutation) patients. iPSC lines were differentiated into neuronal precursors (iPSC-NPCs) and neurons that were subjected to amyloid beta (Aβ) toxicity assays. We found that neurons derived from the fAD patient have a higher susceptibility to Aβ1-42 oligomers compared with neurons coming from healthy and sAD individuals. Our findings suggest that neurons from patients with PSEN1-A246E mutation have intrinsic properties that make them more susceptible to the toxic effects of Aβ1-42 oligomers in the AD brain.

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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Research articleIncreased susceptibility to Aβ toxicity in neuronal cultures derived from familial Alzheimer’s disease (PSEN1-A246E) induced pluripotent stem cells

Highlights

Abstract

Introduction

Section snippets

Patients and fibroblast culture

Generation of iPSCs, iPSC-NPCs and neurons

Discussion

Conclusions

Acknowledgements

Neuron

Clin. Chim. Acta

J. Biol. Chem.

Neurochem. Int.

Lancet

J. Biol. Chem.

Trends Neurosci.

Cell Stem Cell

J. Biol. Chem.

Am. J. Pathol.

Neuropharmacology

Neurobiol. Dis.

Cell

Cell

Neurobiol. Dis.

Cell

Stem Cell Res.

Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease

Antioxid. Redox Signal.

Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders

Annu. Rev. Neurosci.

Protein misfolding, functional amyloid, and human disease

Annu. Rev. Biochem.

Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N-methyl-d-aspartate receptor in mature hippocampal cultures treated with amyloid-beta oligomers

Aging Cell

Soluble protein oligomers as emerging toxins in Alzheimer’s and other amyloid diseases

IUBMB Life

Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide

Nat. Rev. Mol. Cell Biol.

Alzheimer disease in the United States (2010–2050) estimated using the 2010 census

Neurology

Abeta promotes Alzheimer’s disease-like cytoskeleton abnormalities with consequences to APP processing in neurons

J. Neurochem.

Modeling human neurological disorders with induced pluripotent stem cells

J. Neurochem.

Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells

Nature

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response

Nat. Cell Biol.

Molecular mechanisms of amyloid oligomers toxicity

J. Alzheimers Dis.

Research article
Increased susceptibility to Aβ toxicity in neuronal cultures derived from familial Alzheimer’s disease (PSEN1-A246E) induced pluripotent stem cells