Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome.

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase-Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS-specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)-responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

Generation of the Human Pluripotent Stem-Cell-Derived Astrocyte Model with Forebrain Identity.

Astrocytes form functionally and morphologically distinct populations of cells with brain-region-specific properties. Human pluripotent stem cells (hPSCs) offer possibilities to generate astroglia for studies investigating mechanisms governing the emergence of astrocytic diversity. We established a method to generate human astrocytes from hPSCs with forebrain patterning and final specification with ciliary neurotrophic factor (CNTF). Transcriptome profiling and gene enrichment analysis monitored the sequential expression of genes determining astrocyte differentiation and confirmed activation of forebrain differentiation pathways at Day 30 (D30) and D60 of differentiation in vitro. More than 90% of astrocytes aged D95 in vitro co-expressed the astrocytic markers glial fibrillary acidic protein (GFAP) and S100ß. Intracellular calcium responses to ATP indicated differentiation of the functional astrocyte population with constitutive monocyte chemoattractant protein-1 (MCP-1/CCL2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) expression. The method was reproducible across several hPSC lines, and the data demonstrated the usefulness of forebrain astrocyte modeling in research investigating forebrain pathology.

Urine microRNA Profiling Displays miR-125a Dysregulation in Children with Fragile X Syndrome.

A triplet repeat expansion leading to transcriptional silencing of the FMR1 gene results in fragile X syndrome (FXS), which is a common cause of inherited intellectual disability and autism. Phenotypic variation requires personalized treatment approaches and hampers clinical trials in FXS. We searched for microRNA (miRNA) biomarkers for FXS using deep sequencing of urine and identified 28 differentially regulated miRNAs when 219 reliably identified miRNAs were compared in dizygotic twin boys who shared the same environment, but one had an FXS full mutation, and the other carried a premutation allele. The largest increase was found in miR-125a in the FXS sample, and the miR-125a levels were increased in two independent sets of urine samples from a total of 19 FXS children. Urine miR-125a levels appeared to increase with age in control subjects, but varied widely in FXS subjects. Should the results be generalized, it could suggest that two FXS subgroups existed. Predicted gene targets of the differentially regulated miRNAs are involved in molecular pathways that regulate developmental processes, homeostasis, and neuronal function. Regulation of miR-125a has been associated with type I metabotropic glutamate receptor signaling (mGluR), which has been explored as a treatment target for FXS, reinforcing the possibility that urine miR-125a may provide a novel biomarker for FXS.