SummaryDifferentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4–7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

SUMMARYHow mutations in glial fibrillary acidic protein (GFAP) cause Alexander disease (AxD) remains elusive. We generated iPSCs from two AxD patients and corrected the GFAP mutations to examine the effects of mutant GFAP on human astrocytes. AxD astrocytes displayed GFAP aggregates, recapitulating the pathological hallmark of AxD. RNA sequencing implicated the endoplasmic reticulum, vesicle regulation, and cellular metabolism. Corroborating this analysis, we observed enlarged and heterogeneous morphology coupled with perinuclear localization of endoplasmic reticulum and lysosomes in AxD astrocytes. Functionally, AxD astrocytes showed impaired extracellular ATP release, which is responsible for attenuated calcium wave propagation. These results reveal that AxD-causing mutations in GFAP disrupt intracellular vesicle regulation and impair astrocyte secretion, resulting in astrocyte dysfunction and AxD pathogenesis.

Rationale Gaps in our understanding of glutamatergic signaling may be key obstacles in accurately modeling complex CNS diseases. System xc− is an example of a poorly understood component of glutamate homeostasis that has the potential to contribute to CNS diseases. Objectives To determine whether system xc− contributes to behaviors used to model features of CNS disease states. Methods In situ hybridization was used to map mRNA expression of xCT throughout the brain. Microdialysis in the prefrontal cortex was used to sample extracellular glutamate levels; HPLC was used to measure extracellular glutamate and tissue glutathione concentrations. Acute administration of sulfasalazine (8–16 mg/kg, IP) was used to decrease system xc− activity. Behavior was measured using attentional set shifting, elevated plus maze, open-field maze, Porsolt swim test, and social interaction paradigm. Results The expression of xCT mRNA was detected throughout the brain, with high expression in several structures including the basolateral amygdala and prefrontal cortex. Doses of sulfasalazine that produced a reduction in extracellular glutamate levels were identified and subsequently used in the behavioral experiments. Sulfasalazine impaired performance in attentional set shifting, reduced the amount of time spent in an open arm of an elevated plus maze and the center of an open-field maze without altering behavior in a Porsolt swim test, total distance moved in an open-field maze, or social interaction. Conclusions The widespread distribution of system xc− and involvement in a growing list of behaviors suggests that this form of nonvesicular glutamate release is a key component of excitatory signaling.

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Rationale Aspects of schizophrenia, including deficits in sensorimotor gating, have been linked to glutamate dysfunction and/or oxidative stress in the prefrontal cortex. System xc−, a cystine- glutamate antiporter, is a poorly understood mechanism that contributes to both cellular antioxidant capacity and glutamate homeostasis. Objectives Our goal was to determine whether increased system xc− activity within the prefrontal cortex would normalize a rodent measure of sensorimotor gating. Methods In situ hybridization was used to map mRNA expression of xCT, the active subunit of system xc−, in the prefrontal cortex. Prepulse inhibition was used to measure sensorimotor gating; deficits in prepulse inhibition were produced using phencyclidine (0.3–3 mg/kg, sc). N-acetylcysteine (10–100 μM) and the system xc− inhibitor (S)-4-carboxyphenylglycine (CPG, 0.5 μM) were used to increase and decrease system xc− activity, respectively. The uptake of 14C-cystine into tissue punches obtained from the prefrontal cortex was used to assay system xc− activity. Results The expression of xCT mRNA in the prefrontal cortex was most prominent in a lateral band spanning primarily the prelimbic cortex. Although phencyclidine did not alter the uptake of 14C-cystine in prefrontal cortical tissue punches, intra-prefrontal cortical infusion of N-acetylcysteine (10–100 μM) significantly reduced phencyclidine- (1.5 mg/kg, sc) induced deficits in prepulse inhibition. N-acetylcysteine was without effect when co-infused with CPG (0.5 μM), indicating an involvement of system xc−. Conclusions These results indicate that phencyclidine disrupts sensorimotor gating through system xc− independent mechanisms, but that increasing cystine-glutamate exchange in the prefrontal cortex is sufficient to reduce behavioral deficits produced by phencyclidine.