FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice

Abstract

We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.aging | reprogramming | induced neuronal cells R ecent advances in direct reprogramming have demonstrated the feasibility of converting fibroblasts and other terminally differentiated lineages into induced neuronal (iN) cells (1-8). iN cells display elaborate morphologies typical of neurons, express pan-neuronal markers, exhibit hallmarks of functional neurons in that they fire action potentials and form abundant synapses, and, thus, provide a great opportunity for studying the cellular phenotypes of disease-associated genetic mutations (9). To date, most studies used embryonic or early postnatal fibroblasts for reprogramming purposes, but for clinical applications, and in particular for modeling age-related neurodegenerative diseases, it would be preferable to generate iN cells from adult and aged animals.Lineage conversion from aged cells is often challenging, probably due to epigenetic changes during aging. For example, aging has been shown to impair conversion of somatic cells into induced pluripotent stem cells (3,(10)(11)(12). A recent elegant paper demonstrated successful conversion of fibroblasts from old individuals, but cellular properties were not compared between different age groups (13). In embryonic fibroblasts, overexpression of three transcription factors, Brn2, Ascl1, and Myt1l (BAM), efficiently generates iN cells with mature, functional properties within 2 wk (1). Genomic studies and chromatin characterization experiments of fibroblasts shortly after BAM factor expression revealed unique pioneer factor properties of Ascl1, which led to our observation that Ascl1 alone is sufficient to generate functional iN cells, albeit slower and with lower efficiency tha...

Metformin induces FOXO3-dependent fetal hemoglobin production in human primary erythroid cells

“…Our analysis found that individuals with nonsynonymous variants in FOXO3 had lower HbF levels. FOXO3 is a transcription factor that has been shown to be essential for erythroid maturation in mouse models, 22,24 and it is an important regulator of glucose metabolism, 17,60,61 among other functions 14,[62][63][64][65][66] ; however, it has not previously been implicated in HbF regulation. Here, we presented functional studies supporting the hypothesis that FOXO3 is a partial positive regulator of HbF, likely along with other factors; knockdown of FOXO3 in HSPCs from normal individuals reduced g-globin expression and production without altering b-globin expression and production.…”

Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a

“…GFP-tagged miNSCs or hiNSCs (1 × 10 5 ) were plated on the glial cell feeder with NSC medium in a well of 24-well plate. To optimize the differentiation procedure, 24 h before plating miNSCs or hiNSCs, 3 × 10 4 glial cells were plated as the feeder, which were dissociated from P0 C57BL/6J mouse brain as previous described 80 . After 24 h, the medium was replaced with neuronal differentiation medium 1 (N3 medium supplemented with 2% B27, 1% N2, 2 mM glutamax, and 1 × Pen/Strep), which was replaced with neuronal differentiation medium 2 [neurobasal-a medium supplemented with 1 × Insulin-transferrin-selenium solution (Thermo Fisher), 30 ng/ml BDNF, 30 ng/ml CNTF, 30 ng/ml Nerve growth factor (Pepro Tech), 10 μM forskolin, 25 mM l -glutamic acid, 200 mM l -glutamine, 1% B27, 1% N2, and 1 × Pen/Strep].…”

Next‐generation disease modeling with direct conversion: a new path to old neurons

“…Further, in some iN studies, the age of the human donor has been negatively correlated with the percentage of iNs obtained [38], and fibroblasts from adult human donors are resistant to Ascl1/Brn2-based conversion, while fetal fibroblasts are highly amenable [38]. RE1-silencing complex (REST), a major neuronal gene repressor in non-neuronal cells, and the aging-associated TF FOXO3 play important roles in controlling neuronal gene expression and show differential activity between fetal and adult/old fibroblasts, resulting in decreased conversion efficacy in aged starting cells [38][39][40]. Again, the combination of the two pioneer factors Ascl1 and Ngn2, for example, fused via a 2A peptide sequence, has yielded iN efficiencies of typically over 50% across large sample sizes and does not appear to be affected by donor age [9,10,35].…”