Graphical abstract

The pathological potential of human astroglia in Alzheimer's disease (AD) was analysed in vitro using induced pluripotent stem cell (iPSC) technology. Here, we report development of a human iPSC-derived astrocyte model created from healthy individuals and patients with either early-onset familial AD (FAD) or the late-onset sporadic form of AD (SAD). Our chemically defined and highly efficient model provides >95% homogeneous populations of human astrocytes within 30 days of differentiation from cortical neural progenitor cells (NPCs). All astrocytes expressed functional markers including glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-1 (EAAT1), S100B and glutamine synthetase (GS) comparable to that of adult astrocytes in vivo. However, induced astrocytes derived from both SAD and FAD patients exhibit a pronounced pathological phenotype, with a significantly less complex morphological appearance, overall atrophic profiles and abnormal localisation of key functional astroglial markers. Furthermore, NPCs derived from identical patients did not show any differences, therefore, validating that remodelled astroglia are not as a result of defective neural intermediates. This work not only presents a novel model to study the mechanisms of human astrocytes in vitro, but also provides an ideal platform for further interrogation of early astroglial cell autonomous events in AD and the possibility of identification of novel therapeutic targets for the treatment of AD.

EXPERIMENTAL PROCEDURESMaterials-Fura-2/AM was from Molecular Probes, Inc. (Invitrogen). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Dako (Ely, Cambridgeshire, UK). Unless stated otherwise, all other chemicals were purchased from Sigma-Aldrich (Poole, Dorset, UK).Cell Culture-HEK-293 cells, stably transfected with human parathyroid CaR (11), were a gift from Dr. E. F. Nemeth (NPS Pharmaceuticals). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and 200 g/ml hygromycin B (Boehringer-Mannheim, Lewes, Sussex, UK).CaR Phosphorylation Assays-Cells were grown to 80 -90% confluence in 35 mm culture dishes, and CaR T888 phosphorylation was assayed as described previously (15)

The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pH o ) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pH o can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pH o from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca 2+ i ) mobilization, whereas raising pH o to 7.6 potentiated responsiveness to extracellular calcium (Ca i mobilization. Intracellular pH was unaffected by acute 0.4-unit pH o changes, and the presence of physiologic albumin concentrations failed to attenuate the pH o -mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pH o sensitivity. Finally, pathophysiologic pH o elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pH o changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.