In recent years, researchers have honed the craft of transforming skin cells directly into neurons. Now it turns out that these neurons have an advantage over their better-known stem-cell-derived counterparts, according to a study led by Fred Gage at the Salk Institute in La Jolla, California. Directly induced neurons reflect the age of their donors, and even whether they came from a person who has Alzheimer's disease. Though the skin fibroblasts were spared the degenerative disease that was raging in the brain, neurons induced from them adopted an AD signature. Their neuronal identity was shaky, their synaptic function weak, and they showed signs of stress. This signature may have been passed on via epigenetic modifications. In contrast, neurons derived from induced pluripotent stem cells (iPSCs) showed a much milder disease signature. The findings, published April 27 in Cell Stem Cell, suggest that induced neurons may make more accurate models of AD than iPSC-derived ones.
- Transcriptome of induced neurons matches that seen in AD.
- Induced neurons from people with AD are immature, have few synapses.
- These iNs reflect the age of their donors; iPSC-derived neurons do not.
IPSC-derived neurons have become the workhorses for investigating disease mechanisms at the cellular level. However, because they make a pit stop in the embryonic state before differentiating into neurons, iPSC-derived cells are imbued with the characteristics of young cells. Alternatives are neurons induced directly from another differentiated cell type. By using the just-right mix of transcription factors, researchers can transform fibroblasts straight into neurons, bypassing the pluripotent step (Jan 2010 news; Herdy et al., 2019).
Previously, Gage and colleagues had reported that unlike iPSC-derived neurons, which possess attributes of newborn neurons, induced neurons express a transcriptome that reflects the age of the donors (Oct 2015 news). Might they also bear the stamp of neurodegenerative disease?
Finally, LOAD Neurons? Starting with skin biopsies from AD patients and controls, fibroblasts are transformed into neurons. Compared to control iNs, AD iNs have fewer synapses but more stress and cell cycle gene expression. They seem de-differentiated, with open chromatin. iPSC-derived neurons show few signs of disease. [Courtesy of Mertens et al., Cell Stem Cell, 2021.]
To address this question, first author Jerome Mertens and colleagues generated induced neurons (iNs) from fibroblasts of 16 people with AD and of 19 age-matched, non-demented controls per neuropsychological testing and MRI. Then they sequenced both the fibroblast and the iN transcriptomes. The fibroblasts that came from people with AD and controls had no significant differences. However, after conversion to neurons, 778 genes were differentially expressed between the two groups. What's more, this iN AD signature overlapped extensively with gene-expression profiles of neurons plucked directly from postmortem brain samples of people with AD. Genes involved in synaptic transmission were turned down, while stress response and cell cycle re-entry genes were cranked up. “These data indicate that known characteristics of the disease, including functional neuronal failure, stress response, and cell cycle re-entry, are reflected in AD iNs,” the authors wrote.
The donor's disease state was also borne out by functional deficits seen in the iNs. Compared to iNs from controls, those from people with AD had fewer synapses, fired them less frequently, pumped out more reactive oxygen species, and had signs of DNA damage. Curiously, they appeared to take on a hypo-mature state in that their gene-expression pattern suggested a de-differentiation back toward a stem-cell fate. The latter finding jibes with prior studies suggesting the same happens to neurons in the AD brain (Arendt, 2012).
How was this AD signature imparted to fibroblasts, then awakened in neurons? Suspecting epigenetic modifications, the researchers investigated chromatin. They discovered vast stretches of open chromatin in AD iNs, as if transcriptional control was loosening globally. They identified 7,188 stretches of the genome that were differentially accessible in AD iNs compared to control iNs. Of these, 97 percent were more open in AD neurons. The authors believe this epigenetic erosion could explain the fading neuronal identity of AD iNs relative to control iNs. What causes the erosion is unclear, but tau pathology in the AD brain has been tied to a loosening of chromatin (Apr 2017 conference news).
Do iPSC-derived neurons share these characteristics? To investigate, the scientists prepared iPSC-derived neurons from a subset of 20 of their iN donors. They found no significant gene expression differences between iPSC-derived neurons from people with AD versus those from controls. Some gene expression differences nearly reached significance; they largely overlapped with the AD genes identified in directly induced neurons, suggesting a dampened signature.
Since age is the strongest non-genetic risk factor for AD, the researchers asked if the iNs' AD signature reflected a state of accelerated aging. They used several gauges of age, including DNA methylation patterns and expression of age-related genes. As expected, iNs closely aligned with adult neurons, while iPSC-derived neurons resembled fetal cells. Interestingly, the researchers detected no difference in aging markers between AD and control iNs, suggesting that AD is not merely a state of accelerated aging. Rather, AD signatures are distinct from normal aging, but dependent on age.
“The work provides a great model to study the link between aging and neurodegeneration,” commented Bart De Strooper of KU Leuven, Belgium. He thinks the age-related erosion of epigenetic control might be important in AD. “It turns the reasoning in the field upside down, suggesting that the pathological phenotype in AD is a consequence of pathological aging manifested as epigenetic alteration," he wrote to Alzforum (comment below).
De Strooper wondered how the defining neuropathological signature of AD relates to the pathological transcriptional signatures that cropped up in iNs. “The fact that the latter are captured in neurons derived from fibroblasts which were not exposed to amyloid or tangle pathology suggests a cell-autonomous, upstream effect,” he wrote. “How these then affect mechanisms that result in the classical neuropathology remains to be seen. “
The findings raise the question of whether directly induced neurons are better suited to model AD and other diseases of aging than their iPSC-derived counterparts. IPSCs are becoming widely available via a growing repository of isogenic cell lines that can be transformed into different neural cell types. The human induced pluripotent stem cell Neurodegenerative Disease Initiative (iNDI), led by Michael Ward at the National Institute of Neurological Disorders and Stroke and Mark Cookson at the National Institute on Aging, both in Bethesda, Maryland, will introduce 134 disease associated mutations into iPSC lines (May 2021 news). Because fibroblasts are recalcitrant to genome engineering and grow slowly, iNs are less well-suited for this purpose. In a comment to Alzforum, Cookson, Ward, and Andy Singleton of the NIA noted that while iNs may be uniquely positioned to detect signatures of sporadic AD, iPSC-derived neurons harboring familial AD mutations do have transcriptional abnormalities (Aug 2019 news; Feb 2021 news). “For these reasons, our strategy in iNDI complements experiments performed in fibroblast-derived neurons,” they wrote (comment below).—Jessica Shugart
- Research Brief: From Fibroblast to Neuron in One Easy Step
- Neurons Derived Directly from Skin Cells Act the Age of Their Donors
- Location, Conformation, Decoration: Tau Biology Dazzles at AD/PD
- iNDI Aims to Standardize Human Stem Cell Research
- Familial AD Mutations, β-CTF, Spell Trouble for Endosomes
- In Triculture Model, Astrocyte-Microglia Cross Talk Spurs Inflammation
- Herdy J, Schafer S, Kim Y, Ansari Z, Zangwill D, Ku M, Paquola A, Lee H, Mertens J, Gage FH. Chemical modulation of transcriptionally enriched signaling pathways to optimize the conversion of fibroblasts into neurons. Elife. 2019 May 17;8 PubMed.
- Arendt T. Cell Cycle Activation and Aneuploid Neurons in Alzheimer's Disease. Mol Neurobiol. 2012 Apr 13; PubMed.
- Mertens J, Herdy JR, Traxler L, Schafer ST, Schlachetzki JC, Böhnke L, Reid DA, Lee H, Zangwill D, Fernandes DP, Agarwal RK, Lucciola R, Zhou-Yang L, Karbacher L, Edenhofer F, Stern S, Horvath S, Paquola AC, Glass CK, Yuan SH, Ku M, Szücs A, Goldstein LS, Galasko D, Gage FH. Age-dependent instability of mature neuronal fate in induced neurons from Alzheimer's patients. Cell Stem Cell. 2021 Sep 2;28(9):1533-1548.e6. Epub 2021 Apr 27 PubMed.