ABSTRACT
The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD.
Subject(s)
Acetylcholine/physiology , Autism Spectrum Disorder/physiopathology , Craniofacial Abnormalities/physiopathology , Heart Defects, Congenital/physiopathology , Intellectual Disability/physiopathology , Neural Stem Cells/physiology , Neurites/pathology , Acetylcholine/administration & dosage , Autism Spectrum Disorder/complications , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/pathology , Calcium Signaling , Cell Differentiation , Cells, Cultured , Child , Chromosome Deletion , Chromosomes, Human, Pair 9/genetics , Craniofacial Abnormalities/complications , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/pathology , Female , Gene Expression , Heart Defects, Congenital/complications , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Histone-Lysine N-Methyltransferase/genetics , Humans , Intellectual Disability/complications , Intellectual Disability/genetics , Intellectual Disability/pathology , Male , Models, Neurological , Mutation , Pluripotent Stem Cells/physiology , RNA, Messenger/metabolismABSTRACT
During their phase of developmental programmed cell death (PCD), neurons depend on target-released trophic factors for survival. After this period, however, they critically change as their survival becomes target-independent. The molecular mechanisms underlying this major transition remain poorly understood. Here, we investigated, which transcription factors (TFs) might be responsible for the closure of PCD. We used Purkinje cells as a model since their PCD is restricted to the first postnatal week in the mouse cerebellum. Transcriptome analysis of Purkinje cells during or after PCD allowed the identification of Krüppel like factor 9 (Klf9) as a candidate for PCD closure, given its high increase of expression at the end of the 1st postnatal week. Klf9 function was tested in organotypic cultures, through lentiviral vector-mediated manipulation of Klf9 expression. In absence of trophic factors, the Purkinje cell survival rate is of 40%. Overexpression of Klf9 during PCD dramatically increases the Purkinje cell survival rate from 40% to 88%, whereas its down-regulation decreases it to 14%. Accordingly, in organotypic cultures of Klf9 knockout animals, Purkinje cell survival rate is reduced by half as compared to wild-type mice. Furthermore, the absence of Klf9 could be rescued by Purkinje cell trophic factors, Insulin growth factor-1 and Neurotrophin3. Altogether, our results ascribe a clear role of Klf9 in Purkinje cell survival. Thus, we propose that Klf9 might be a key molecule involved in turning off the phase of Purkinje PCD.
Subject(s)
Kruppel-Like Transcription Factors/genetics , Purkinje Cells/metabolism , Animals , Cell Death/drug effects , Cell Death/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cerebellum/cytology , Cerebellum/metabolism , Insulin-Like Growth Factor I/pharmacology , Kruppel-Like Transcription Factors/metabolism , Mice , Mice, Knockout , Neurotrophin 3/pharmacology , Organ Culture Techniques , Purkinje Cells/physiology , Transcription Factors/metabolism , Transcription, Genetic , TranscriptomeABSTRACT
The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.