Scientists have used induced pluripotent stem cell (iPSC)-derived neurons generated from Alzheimer disease (AD) patients’ skin cells to characterize familial and sporadic forms of the disease. A team led by researchers at the Howard Hughes Medical Institute says their results indicate that sporadic AD may actually be subdivisible into different categories, dependent on whether it’s the neurons themselves or other cell types, such as astrocytes, that are altered.
Reporting in Nature, Lawrence S. B. Goldstein, Ph.D., and colleagues, claim that their work in addition demonstrates how iPSC technology can be used to study AD phenotypes, even though it can take decades for the disease to become overtly manifest in patients. Their published paper is titled “Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells.”
AD is characterized post mortem by the presence of both extracellular amyloid plaques composed of amyloid-β peptides, and neurofibrillary tangles made up of intraneuronal aggregations of hyperphosphorylated tau, a microtubule-associated protein involved in microtubule stabilization. However, the authors note, “the causative relationship between amyloid plaque/amyloid-β and tau pathologies is unclear in humans.”
The vast majority of AD cases occur as sporadic incidences, but most analyses of AD at the cellular level in animal models and humans has centered on the rare, dominantly inherited familial forms of the disease, which feature mutations or duplications in either the amyloid-β precursor protein (APP) gene, or the presenilin genes. And even this work hasn’t been able to definitively demonstrate whether physiologically relevant levels of amyloid-β directly cause phosphorylated tau (p-tau), Dr. Goldstein et al point out.
The Howard Hughes team set out to generate neurons from iPSCs derived from skin cell fibroblasts taken from patients with both sporadic AD (sAD) and familial AD (fAD), to see whether this approach could be used to observe AD phenotype, and determine whether there is a causal relationship between APP processing and tau phosphorylation. Another question to answer was whether neurons with the genome of sAD patients would exhibit the same phenotype as those from patients with familial AD.
To this end, the researchers generated iPSC lines, and then neurons, from primary fibroblasts from two patients with sporadic AD (sAD1 and sAD2), and two patients with familial disease, both of which were caused by a duplication of the amyloid-β precursor protein (APPDp). Neurons were similarly derived from two control individuals without dementia (nondemented control, or NDC). The methodology is described in the paper.
These iPSC-derived neurons were then purified to near homogeneity, and their passive membrane properties and ability to generate voltage-dependent action potentials, currents, and functional synaptic contacts, confirmed through multiple electrophysiological studies. Protein expression analysis indicated that both glutamatergic and GABAergic neuronal subtypes were present. “Importantly, no significant differences in neuronal subtypes were detected between patients and controls,” the researchers state.
Analyses of protein expression in the resulting neurons confirmed that those derived from patients APPDp1, APPDp2, and sAD2 secreted significantly higher levels of amyloid-β compared with the NDC neurons. Similarly, neurons from both the APPDp patients and sAD2 also demonstrated significantly higher p-tau/total tau ratios, and upregulation of aGSK-3β activity than NDC-derived neurons. Neurons derived from patient sAD1 didn’t demonstrate raised amyloid-β or p-tau levels.
Given that for neurons from three of the four patients there was a correlation between amyloid-β, p-tau/total tau, and aGSK-3β levels, the researchers postulated that if APP proteolytic products such as amyloid-β or carboxy-terminal fragments (CTFs) play a causal role in p-tau and aGSK-3β elevation, then inhibiting secretase activity would act to reduce p-tau and aGSK-3β. In fact, they found that both the p-tau/total tau ratio and aGSK-3β levels could be partly normalized in neurons from sAD2 and the APPDp patients, using treatment with a β-secretase inhibitor. This supports the notion that the APP processing pathway has a causative role in tau phosphorylation in human neurons, the investigators write.
The finding that purified neurons derived from patient sAD1 fibroblasts didn’t demonstrate a significant AD phenotype is particularly important, they add. It indicates that sporadic AD may have two forms of genetic basis, one involving changes in the neurons themselves, and the other that results in alterations in other cell types such as astrocytes.
“Thus, future iPSC studies examining larger numbers of patients and controls have the potential to provide great insight into the mechanisms behind the observed heterogeneity in sporadic Alzheimer disease pathogenesis, the role of different cell types, patient-specific drug responses, and prospective diagnostics.”