SummaryTo determine the role for mutations of MECP2 in Rett syndrome, we generated isogenic lines of human induced pluripotent stem cells, neural progenitor cells, and neurons from patient fibroblasts with and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal-specific gene expression changes, including induction of a senescence-associated secretory phenotype (SASP) program. Patient-derived neurons made without MECP2 showed signs of stress, including induction of P53, and senescence. The induction of P53 appeared to affect dendritic branching in Rett neurons, as P53 inhibition restored dendritic complexity. The induction of P53 targets was also detectable in analyses of human Rett patient brain, suggesting that this disease-in-a-dish model can provide relevant insights into the human disorder.
27To determine the role for mutations of MECP2 in Rett Syndrome, we generated isogenic 28 lines of human iPSCs (hiPSCs), neural progenitor cells (NPCs), and neurons from patient 29 fibroblasts with and without MECP2 expression in an attempt to recapitulate disease 30 phenotypes in vitro. Molecular profiling uncovered neuronal specific gene expression 31 changes including induction of a Senescence Associated Secretory Phenotype (SASP) 32 program. Patient derived Neurons made without MECP2 show signs of stress, including 33 induction of p53, and senescence. The induction of p53 appeared to affect dendritic 34 branching in Rett neurons, as p53 inhibition restored dendritic complexity. These 35 disease-in-a-dish data suggest that loss of MECP2 can lead to dendritic defects due to 36 an increase in aspects of neuronal aging. 37 38 39 40 41 42 43 44 45 46Rett Syndrome is a disease associated with loss of function mutations in the gene 48 MECP2, which was originally identified as encoding a methylated DNA binding protein 1-49 3 . Patient symptoms include microcephaly, intellectual disability, facial dysmorphia, and 50 seizure activity 4,5 . Studies in murine models recapitulate many of the patient phenotypes 51 and have recently identified a role for MECP2 particularly in inhibitory neurons [6][7][8][9] . These 52 studies demonstrated that loss of MECP2 can lead to defects in transcription 10-12 , 53 dendritic branching 13 , nuclear size 3 , and AKT signaling 14 .54 55 MECP2 has also been described as a transcription factor with specific targets 10,11,13 , and 56 more broadly as either a transcriptional activator 14 or repressor [15][16][17][18] . However, despite 57 decades of research on MECP2, it is still unclear how mutations in this protein lead to 58 patient symptoms 3,14,[19][20][21] . To confirm findings made in other models and further study 59 these in a human system, some have turned to modeling Rett Syndrome in vitro by taking 60 advantage of Disease in a dish approaches. This involves making hiPSCs from patient 61 somatic cells, or using genome engineering to introduce mutations into WT human 62 pluripotent stem cells. In either case, the pluripotent stem cells created are then 63 differentiated toward the neural lineage, and then comparisons can be made between 64 cells that express MECP2 or lack it. 65 66 Some of these studies have even taken advantage of isogenic or congenic cells lines to 67 identify both transcriptional and electrophysiological effects of loss of MECP2 in human 68 4 in vitro models 14,22 . In the current study, we also sought to mitigate the effect of genetic 69 background and variability of differentiation by taking advantage of several congenic lines 70 of hiPSCs that either express the WT allele or the mutant allele leading to cells that 71 express or lack MECP2 23 . This allowed for detailed molecular analyses of hiPSCs, NPCs 72 and neurons with and without MECP2 under the same genetic background. In addition, 73 several lines were made and analyzed in each category to avoid vari...
Rett Syndrome is characterized by a postnatal loss of neurophysiological function and regression of childhood development. Because the syndrome is X-linked and males with MECP2 mutations generally do not survive birth, the study of this syndrome has been complicated by the fact that in female brain, a portion of neurons express wild type MECP2, and another portion express a non-functional allele of MECP2. Therefore, bulk-RNA-sequencing of Rett brain is confounded by the presence of chimerism of neurons for functional MECP2 in neurons. We developed an approach that allows for single-nuclei transcriptional profiling of individual neurons and a direct comparison between neurons that express functional MECP2 with those that express the disease-causing allele. We found that mutant neurons from Rett brain show patterns of aberrant expression of synaptic and metabolic genes, both of which can be detected in in vitro models of Rett Syndrome. We used these resources to identify a role for POU2F1/OCT1 transcription factor in mediating the response to stress due to loss of MECP2, highlighting a potential key molecular regulator of stress in Rett neurons. Together, our new sorting approach enables us to highlight defective molecular and metabolic pathways in Rett brain neurons and suggests that in vitro models could serve as valuable tools to further study this syndrome and potentially for development of novel therapeutics.
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