title: Limitations of In Vivo Reprogramming to Dopaminergic Neurons via a Tricistronic Strategy.
abstract: Parkinson's disease is one of the most common neurodegenerative disorders characterized by cell death of dopaminergic neurons in the substantia nigra. Recent research has focused on cellular replacement through lineage reprogramming as a potential therapeutic strategy. This study sought to use genetics to define somatic cell types in vivo amenable to reprogramming. To stimulate in vivo reprogramming to dopaminergic neurons, we generated a Rosa26 knock-in mouse line conditionally overexpressing Mash1, Lmx1a, and Nurr1. These proteins are characterized by their role in neuronal commitment and development of midbrain dopaminergic neurons and have previously been shown to convert fibroblasts to dopaminergic neurons in vitro. We show that a tricistronic construct containing these transcription factors can reprogram astrocytes and fibroblasts in vitro. However, cassette overexpression triggered cell death in vivo, in part through endoplasmic reticulum stress, while we also detected "uncleaved" forms of the polyprotein, suggesting poor "cleavage" efficiency of the 2A peptides. Based on our results, the cassette overexpression induced apoptosis and precluded reprogramming in our mouse model. Therefore, we suggest that alternatives must be explored to balance construct design with efficacious reprogramming. It is evident that there are still biological obstacles to overcome for in vivo reprogramming to dopaminergic neurons.
authors.affiliation: 1 Institute of Developmental Genetics , Helmholtz Zentrum München, Neuherberg, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Theodorou
authors.affiliation: 1 Institute of Developmental Genetics , Helmholtz Zentrum München, Neuherberg, Germany.
authors.forename: Benedict
authors.initials: B
authors.lastname: Rauser
authors.affiliation: 1 Institute of Developmental Genetics , Helmholtz Zentrum München, Neuherberg, Germany.
authors.forename: Jingzhong
authors.initials: J
authors.lastname: Zhang
authors.affiliation: 1 Institute of Developmental Genetics , Helmholtz Zentrum München, Neuherberg, Germany.
authors.forename: Nilima
authors.initials: N
authors.lastname: Prakash
authors.affiliation: 1 Institute of Developmental Genetics , Helmholtz Zentrum München, Neuherberg, Germany.|2 Developmental Genetics c/o Helmholtz Zentrum München, Technische Universität München-Weihenstephan , Neuherberg/Munich, Germany.|3 German Center for Neurodegenerative Diseases (DZNE) , Munich, Germany.|4 Munich Cluster for Systems Neurology (SyNergy), Adolf-Butenandt-Institut Ludwig-Maximilians-Universität München , Munich, Germany .
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wurst
authors.affiliation: 1 Institute of Developmental Genetics , Helmholtz Zentrum München, Neuherberg, Germany.
authors.forename: Joel A
authors.initials: JA
authors.lastname: Schick
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D016923:Cell Death / Q000235:genetics; D002460:Cell Line; D065150:Cellular Reprogramming / Q000235:genetics*; D059290:Dopaminergic Neurons / Q000166:cytology* / Q000378:metabolism*; D053595:Embryonic Stem Cells; D005260:Female; D005347:Fibroblasts; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D059765:Homologous Recombination; D060850:LIM-Homeodomain Proteins / Q000235:genetics; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes; D056811:Unfolded Protein Response / Q000235:genetics
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D060850:LIM-Homeodomain Proteins; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D014157:Transcription Factors; C499317:Ascl1 protein, mouse; C489692:Lmx1a protein, mouse; C539883:Nr4a2 protein, mouse