FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice Henrik Ahleniusa,1,2, Soham Chandaa,b,1, Ashley E. Webbc,3, Issa Yousifa, Jesse Karmazina, Stanley B. Prusinerd,e,f, Anne Brunetc, Thomas C. Südhofb,g,4, and Marius Werniga,4 aInstitute for Stem Cell Biology and Regenerative Medicine and Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305; cDepartment of Genetics, Stanford University School of Medicine, Stanford, CA 94305; dInstitute for Neurodegenerative Diseases, University of California, San Francisco, CA 94158; eDepartment of Neurology, University of California, San Francisco, CA 94158; fDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; and gHoward Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Thomas C. Südhof, June 1, 2016 (sent for review August 24, 2015; reviewed by Anton Maximov, Yi E. Sun, and Li-Huei Tsai) We and others have shown that embryonic and neonatal fibroblasts shares common genomic targets with Ascl1 and, when overex- can be directly converted into induced neuronal (iN) cells with mature pressed, significantly impairs iN cell reprogramming (16). FoxO3 functional properties. Reprogramming of fibroblasts from adult and functions downstream of the insulin/IGF signaling pathway and aged mice, however, has not yet been explored in detail. The ability has also been shown to maintain adult neural precursor cell – to generate fully functional iN cells from aged organisms will be homeostasis (17 19). Thus, FoxO3 might indirectly also affect particularly important for in vitro modeling of diseases of old age. neurogenesis. Here, we demonstrate production of functional iN cells from Our aims for the present study were threefold: first, we wanted fibroblasts that were derived from mice close to the end of their to assess whether it is possible to reprogram fibroblasts from aged mice into neurons; second, we wanted to elucidate whether aging lifespan. iN cells from aged mice had apparently normal active and affects the dynamics of neuronal conversion of fibroblasts; and passive neuronal membrane properties and formed abundant syn- finally, we wanted to investigate whether intrinsic, aging-associated aptic connections. The reprogramming efficiency gradually decreased factors, such as members of the FoxO family of transcription fac- with fibroblasts derived from embryonic and neonatal mice, but tors, influence iN cell formation in an age-dependent manner. remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box Results O3 (FoxO3), which is implicated in aging, blocked iN cell conversion Generation of Functional iN Cells from Fibroblasts of Old Mice. We of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 have shown that forced expression of a pool of three transcription increased the reprogramming efficiency of adult-derived but not of factors, Brn2, Ascl1, and Myt1l, successfully converts mouse em- embryonic fibroblasts and also enhanced functional maturation of bryonic fibroblasts (MEFs) and early postnatal tail-tip fibroblasts resulting iN cells. Hence, FoxO3 has a central role in the neuronal (TTFs) into iN cells (1). To establish whether aging fibroblasts can reprogramming susceptibility of cells, and the importance of FoxO3 also be converted into iN cells, we derived cultures from tails of appears to change during development. 17- to 20-month-old Tau:EGFP mice that express EGFP from the endogenous, neuron-specific Tau locus (20). The tail-tip fibroblasts aging | reprogramming | induced neuronal cells were then lentivirally transduced with BAM transcription factors. ecent advances in direct reprogramming have demonstrated Significance Rthe feasibility of converting fibroblasts and other terminally differentiated lineages into induced neuronal (iN) cells (1–8). iN Direct reprogramming of nonneuronal cells into induced neuronal cells display elaborate morphologies typical of neurons, express (iN) cells has been of great interest for its potential applications in pan-neuronal markers, exhibit hallmarks of functional neurons in neurological disease modeling. Although this approach is well- that they fire action potentials and form abundant synapses, and, established using cells from embryonic and neonatal animals, its thus, provide a great opportunity for studying the cellular pheno- applicability for cells from adult and aged animals has remained types of disease-associated genetic mutations (9). To date, most unexplored. This study demonstrates that iN cells can be effi- studies used embryonic or early postnatal fibroblasts for reprog- ciently generated from aging animals, but that age-dependent ramming purposes, but for clinical applications, and in particular roadblocks which depend on the transcription factor FoxO3 affect for modeling age-related neurodegenerative diseases, it would be the reprogramming efficiency and functional maturation of iN cell preferable to generate iN cells from adult and aged animals. properties. Eliminating these roadblocks may be useful for mod- Lineage conversion from aged cells is often challenging, eling age-related neurological disorders using iN cells. probably due to epigenetic changes during aging. For example, aging has been shown to impair conversion of somatic cells into Author contributions: H.A., S.C., S.B.P., A.B., T.C.S., and M.W. designed research; H.A., S.C., – A.E.W., I.Y., and J.K. performed research; A.B. contributed new reagents/analytic tools; induced pluripotent stem cells (3, 10 12). A recent elegant H.A., S.C., A.E.W., J.K., T.C.S., and M.W. analyzed data; and H.A., S.C., and M.W. wrote paper demonstrated successful conversion of fibroblasts from the paper. old individuals, but cellular properties were not compared Reviewers: A.M., The Scripps Research Institute; Y.E.S., University of California, Los between different age groups (13). In embryonic fibroblasts, Angeles; and L.-H.T., Massachusetts Institute of Technology. overexpression of three transcription factors, Brn2, Ascl1, The authors declare no conflict of interest. and Myt1l (BAM), efficiently generates iN cells with mature, 1H.A. and S.C. contributed equally to this work. functional properties within 2 wk (1). Genomic studies and 2 Present address: Department of Clinical Sciences, Lund Stem Cell Center, University Hospital chromatin characterization experiments of fibroblasts shortly BMC B10, SE-221 84 Lund, Sweden. after BAM factor expression revealed unique pioneer factor 3Present address: Department of Molecular Biology, Cell Biology and Biochemistry, Brown properties of Ascl1, which led to our observation that Ascl1 University, Providence, RI 02903. alone is sufficient to generate functional iN cells, albeit slower 4To whom correspondence may be addressed. Email: [email protected] or wernig@ and with lower efficiency than when all three BAM transcrip- stanford.edu. tion factors are used (14, 15). Recently, we observed that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the longevity-associated forkhead transcription factor, FoxO3, 1073/pnas.1607079113/-/DCSupplemental. 8514–8519 | PNAS | July 26, 2016 | vol. 113 | no. 30 www.pnas.org/cgi/doi/10.1073/pnas.1607079113 + Three weeks later, we detected bright EGFP cells with clear Developmental Stage but Not Adult Aging Impairs iN Cell Reprogramming. neuronal morphologies and expression of the pan-neuronal marker To more systematically evaluate the impact of aging on reprog- Tuj1 (Fig. 1A). Electrophysiological characterization demonstrated ramming efficiency of fibroblasts, we derived TTFs from mice of that TTF-derived iN (TTF iN) cells generated from aged animals different ages including embryonic (MEF), postnatal (4 d), young were functional in terms of firing action potentials (APs) and adult (3 mo), middle aged (10 and 15 mo), and aged (25 mo) mice. exhibiting voltage-gated sodium and potassium currents (Fig. 1B). Following BAM transduction, fibroblasts from all ages were able to Thus, the BAM reprogramming factors are capable of converting form Tuj1 and Map2 immunopositive iN cells with neuronal fibroblasts from aged donors into functional neurons. morphology (Fig. 1C). However, the reprogramming efficiency and A B i ii iii CD NEUROSCIENCE Fig. 1. Fibroblasts from postnatal and adult mice are less susceptible to iN cell reprogramming than MEFs. (A) Experimental approach (Left) and image of iN cells (Right) generated from TTFs of a 17-mo-old (17M) Tau-EGFP animal. Micrograph showing Tau-EGFP epifluorescence (green) and immunofluorescence detection with Tuj1 antibodies (red). (Scale bar, 30 μm.) (B) Sample traces of spontaneous AP firing (i), current-pulse–induced AP (ii), and voltage-gated Na+ + and K currents (iii) recorded from TTF iN cells reprogrammed from a 20-mo-old animal (20M TTF-iN cell). Insets in red, magnified view of corresponding boxed area. (C) Representative images (Left) for Tuj1 (green) and Map2 (red) immunoreactivity of TTF iN cells generated from embryonic (MEF), postnatal (4 d old; 4D), adult (3 mo old; 3M), middle aged (10 and 15 mo old; 10M and 15M, respectively) and aged (25 mo old; 25M) animals, 3 wk after induction. (Scale + + bar, 50 μm.) (D) Average fractions of Tuj1 (Top Right) and Map2 (Bottom Right) cells 3 wk after infection