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PSEN1 Mutant iPSC-Derived Model Reveals Severe Astrocyte Pathology in Alzheimer's Disease

Summary Alzheimer's disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased β-amyloid production, altered cytokine release, and dysregulated Ca2+ homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca2+ transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases.


INTRODUCTION
Alzheimer's disease (AD) is a common dementing disorder characterized by progressive decline of cognitive functions, especially memory loss. The neuropathology of AD includes extracellular deposits of b-amyloid (Ab) in senile plaques, intracellular neurofibrillary tangles comprising hyperphosphorylated tau, synaptic dysfunction, and neuronal death (Blennow et al., 2006). While most AD cases are sporadic late-onset type (LOAD), 1%-2% of the cases are of a familial early-onset type AD (EOAD), with underlying mutations in presenilin-1 and -2 (PSEN1/2) or amyloid precursor protein (APP) genes (Waring and Rosenberg, 2008). Neuropathological changes and apparent cellular dysfunctions are similar in various forms of LOAD and EOAD, but the exact mechanisms underlying the onset and progression of AD are not well understood. According to the largely accepted amyloid cascade hypothesis, the extracellular accumulation of Ab peptides triggers the onset of AD (Hardy and Selkoe, 2002). Although considerable pre-clinical and clinical evidence supports the amyloid cascade model, all experimental therapies built on this hypothesis have thus far been unsuccessful in clinics (Castello et al., 2014;Golde et al., 2011). Several factors have probably contributed to the failures in AD drug development, including unsuitable pre-clinical research models, such as transgenic mice or human tumorderived cell lines, which do not fully recapitulate the human disease. Recent cellular models created from patient cells using induced pluripotent stem cell (iPSC) technology have provided promising tools for understanding human disease mechanisms. So far, most of the human iPSC (hiPSC)-based AD models have concentrated on hippocampal or cortical neurons (Kondo et al., 2013;Nieweg et al., 2015), but other cell types of the CNS are also likely to contribute to AD pathology.
Astrocytes are the most abundant non-neuronal cell type in the CNS and have multiple indispensable tasks in brain development and function, including energy supply to neurons in the form of lactate, as well as synapse formation and maintenance (Belanger et al., 2011;Oberheim et al., 2006). Human astrocytes are about 20 times larger, integrate 20 times more synapses, and propagate Ca 2+ waves far more quickly than their rodent counterparts (Oberheim et al., 2009). Moreover, the greatest genetic difference between human and rodent brain has been identified to be in glial transcripts (Zhang et al., 2016). As astrocytes have been suggested to have a role in AD pathogenesis (Vincent et al., 2010), we generated hiPSC-derived astrocytes from patients with EOAD carrying PSEN1 exon 9 deletion (4.6 kb deletion; the Finnish PSEN1 DE9) (Crook et al., 1998) and report here that astrocytes manifest many hallmarks of AD pathology. Our findings highlight the importance of astrocytes in AD pathology and demonstrate that hiPSC-derived astrocytes provide a valuable tool for studying AD disease mechanisms.

RESULTS
Patient and Control Cells iPSC lines were generated from three individuals carrying the PSEN1 DE9 mutation, two diagnosed with AD and one pre-symptomatic with no clinical diagnosis, and from three healthy adult control individuals (Table 1). All six individuals carried the neutral 33/ 33 isoforms of APOE, the most important risk gene for LOAD. To examine the cause-effect relationship between DE9 mutation and AD phenotype, we also generated gene-corrected isogenic control lines from one symptomatic AD patient and the pre-symptomatic PSEN1 DE9 carrier using a previously published donor plasmid-mediated CRISPR/Cas9 workflow ( Figure S1 and Table 1) (Chen et al., 2015). After 10 passages, all iPSC lines showed typical morphological characteristics of pluripotent stem cells as well as high expression of pluripotency markers (Figures S2A and S2B; Table S1). All the studied lines formed embryoid bodies, differentiated spontaneously toward the three germ layers, and presented normal euploid karyotypes (Figures S2C-S2F and Table S1). Insertion of the exon 9 in the isogenic control lines was confirmed by PCR amplifications of the targeted area (Figures S1D-S1F) and Sanger sequencing across the deletion breakpoints (data not shown).
iPSCs Efficiently Differentiate into Functional Astrocytes Astrocyte differentiation was carried out by a slightly modified protocol from Krencik et al. (2011) (Figure 1A). Ciliary neurotrophic factor (CNTF) and bone morphogenetic protein 4 (BMP4) were applied in the final maturation stage, as they have been shown to promote the generation of bona fide astrocytes (Magistri et al., 2016). The mRNA expression of GFAP increased gradually to about 100-fold from 1-month-old to 6-month-old spheres, and further by 20-fold when maturating the dissociated cells for 7 days ( Figure 1B). At the age of 6 months, no TUJ1-immunoreactive neuronal cells were observed in the dissociated sphere cultures ( Figure 1C). Finally, more than 90% of the cells were GFAP and/or S100b positive, acquiring a stellate morphology, after maturation with CNTF and BMP4 ( Figure 1D). The mRNA expression levels of GFAP and S100B were significantly higher when compared with iPSC-derived neurons from the same lines ( Figure 1E). The matured astrocytes from all the lines, independent of the disease status, showed also other typical characteristics, such as high mRNA expression of the astrocyte-specific glutamate transporters SLC1A2 and SLC1A3, and water channel AQP4 ( Figure 1F) in comparison with neurons. Cells were able to take up glucose, produce and secrete cytokines and glutathione, and propagate intercellular Ca 2+ waves . No evident differences were seen in the differentiation potential between the genotypes, and both AD and control iPSCs generated comparable, functional astrocytes. PSEN1 DE9 Mutant Astrocytes Contribute to b-Amyloid Pathology Wild-type presenilin-1 (PS-1) is initially translated as a 43-kDa molecule, which is processed to stable N-terminal (NTF) and C-terminal fragments (CTF) of 27 kDa and 17 kDa, respectively (Thinakaran et al., 1996). PS-1 NTFs and CTFs are critically involved in composing the active g-secretase complex. Since PSEN1 DE9 mutation leads to an in-frame deletion of the endoproteolytic site and consequently to the accumulation of uncleaved PS-1 of a smaller molecular weight (40 kDa) as compared with the  (C) Representative immunocytochemistry images of cells dissociated from 3-or 6-month-old spheres stained for TUJ1 (green) and GFAP (red). Nuclei are stained with Hoechst. Scale bars, 100 mm.
(D) Representative immunocytochemistry images of astrocytes from control, AD, and isogenic control lines matured with CNTF and BMP4 for 7 days, stained for S100b (green) and GFAP (red). Nuclei are stained with Hoechst. Scale bars, 50 mm.
(legend continued on next page) wild-type PS-1 (Thinakaran et al., 1996), we analyzed PS-1 endoproteolytic cleavage. As expected, PSEN1 DE9 mutant astrocytes showed robust accumulation of full-length PS-1, which was undetectable in control cells, and subsequent reduction in CTFs (Figures 2A and 2B). However, we did not observe differences in the enzymatic activity of g-secretase ( Figure 2G), nor at the protein expression level of APP (Figures 2A and 2C). Neurons have been thought to be the main source of Ab production, but astrocytes are also able to secrete Ab (Liao et al., 2016). Thus, we quantified Ab1-40 and Ab1-42 from the astrocyte culture media. The secretion of Ab1-42 was increased about 5-fold in the AD cultures when compared with the controls, whereas Ab1-40 secretion was not altered ( Figures 2D, 2E, S4A, and S4B). This led to a significantly increased Ab1-42/Ab1-40 ratio and toxic Ab profile in AD astrocytes (Figures 2F and S4C). Treatment with the g-secretase inhibitor DAPT decreased the production of Ab1-40 and completely blocked the production of Ab1-42 in both AD and control astrocytes ( Figures 2D and 2E). The overall Ab production by astrocytes was comparable with that of iPSC-derived neurons from the same lines ( Figures S4D and S4E), and the mRNA expression levels of APP and BACE1 were only slightly lower in astrocytes when compared with neurons ( Figures S4G and S4H), highlighting the importance of astrocytes as Ab-producing cells. Importantly, both the PS-1 endoproteolytic processing and Ab secretion were resolved in the isogenic control lines (Figure 2), verifying that the partial correction of the deletion had restored PS-1 function. We next measured the astrocytic uptake of Ab1-42 by fluorescence-activated cell sorting (FACS). After 16 hr of incubation with a fluorochrome-conjugated fibrilized Ab1-42, internalized Ab was quantified and AD astrocytes showed reduced capacity to take up Ab when compared with controls ( Figure 2H). These results show that PSEN1 DE9 astrocytes present the endoproteolytic defect typical for this specific AD mutation and that AD astrocytes may contribute to amyloid pathology by both increased release and compromised uptake of Ab1-42.

Mutant Astrocytes
Several PSEN1 mutations, including PSEN1 DE9, have been reported to disturb Ca 2+ release from the ER (Cedazo- Minguez et al., 2002;Ito et al., 1994). Thus, we next analyzed ER Ca 2+ cycling and especially Ca 2+ leakage from the ER. The non-specific Ca 2+ leakage was studied by simultaneous blocking of ryanodine receptor (RyR), inositol triphosphate receptor (IP 3 R), and SERCA ( Figure 3A). The rate of the non-specific Ca 2+ release, represented by the slope of the increase in [Ca 2+ ] i level, was faster in AD than in control astrocytes ( Figures 3B and S5). These results show that PSEN1 DE9 AD astrocytes manifest altered cellular Ca 2+ homeostasis.

Cytokine Secretion after Inflammatory Stimulation Is Altered in PSEN1 DE9 Mutant Astrocytes
Given that astrocytes contribute to neuroinflammation in AD (Heneka et al., 2015), we analyzed the cytokine secretion profile following pro-inflammatory stimulation. The optimal stimulation was determined by comparing two key pro-inflammatory mediators increased in AD brain, interleukin-1b (IL-1b) (10 ng/mL) and tumor necrosis factor a (TNFa) (50 ng/mL). Stimulation of control astrocytes with IL-1b and/or TNFa for 48 hr led to increased cytokine secretion to media ( Figure S3A). Concomitantly, the expression of inflammation-related genes IL1B, IL6, IL10, TNF, CCL5, and NOS2 was upregulated after stimulation ( Figure S3B). Importantly, stimulation with lipopolysaccharide had no effect ( Figures S3A and S3B), which is in line with the previous knowledge on human astrocytes and further validates the identity of our cells (Tarassishin et al., 2014). We next treated astrocytes with a combination of IL-1b and TNFa and compared the cytokine secretion between AD and control cells. Upon inflammatory stimulation, PSEN1 DE9 astrocytes secreted significantly higher levels of IL-2, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF) than control astrocytes, whereas secretion of CCL5 was lower in the PSEN1 DE9 cultures ( Figure 4). Interestingly, treatment with g-secretase inhibitor DAPT led to significant reduction in IL-2 and GM-CSF secretion from AD astrocytes while it had no effect on control cells ( Figure 4). These data suggest that the altered cytokine secretion profile of astrocytes may enhance neuroinflammation in AD.

Altered Metabolism in PSEN1 DE9 Mutant Astrocytes Leads to Increased ROS and Reduced Lactate Production
We next analyzed the metabolic activity of the cells by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) by Seahorse XF Technology ( Figures 5A and 5B). Interestingly, PSEN1 DE9 astrocytes were more oxidative than isogenic control cells, which relied more on glycolysis as typical for astrocytes ( Figures  5C-5E). Treatment with the g-secretase inhibitor DAPT had no effect on the metabolism of the astrocytes ( Figures  5C-5E). Since increased oxidative stress has been suggested to play a crucial role in AD pathology (Beal, 2005;Lovell and Markesbery, 2007), we measured cellular oxidative stress using CellROX, a fluorogenic probe, which becomes fluorescent upon oxidation. PSEN1 DE9 astrocytes showed significantly increased levels of intracellular reactive oxygen species (ROS) when compared with isogenic control cells ( Figures 5F and 5G). As the decreased glycolysis also suggested a reduction in lactate production, we further measured L(+)-lactate secretion. As expected, lactate secretion to the media was significantly higher from the control than from the PSEN1 DE9 astrocytes ( Figure 5H). These data show that PSEN1 DE9 astrocytes are more oxidative than and APP (C) levels. Results normalized against GAPDH and shown as percentage of control (CTRL, n = 6 lines; isogenic CTRL, n = 4 replicates from 2 lines; AD, n = 6 lines; ***p < 0.001).
the glycolytic control astrocytes, generating more ROS and oxidative stress, and producing less lactate.

PSEN1 DE9 Mutant Astrocytes Alter the Calcium Signaling Activity of Healthy Neurons
Finally, we established a 3D co-culture model of neurons and astrocytes to study whether PSEN1 DE9 mutant astrocytes have functional effects on neurons. We utilized a previously described (Choi et al., 2014) thin-layer Matrigel model to culture isogenic control neurons together with either isogenic control or PSEN1 DE9 mutant astrocytes ( Figure 6A). Application of glutamate, together with the co-agonist glycine, resulted in significantly lower Ca 2+ -transient amplitudes in healthy control neurons co-cultured with AD astrocytes when compared with the same neurons co-cultured with control astrocytes ( Figures  6B-6D). Likewise, the presence of AD astrocytes also significantly reduced the neuronal Ca 2+ transients evoked by g-aminobutyric acid (GABA) (Figures 6B, 6C, and 6E). These results show that the PSEN1 DE9 astrocytes trigger functional consequences on healthy neurons.

DISCUSSION
Current knowledge of the mechanisms underlying AD pathology mostly arises from animal models, which do not truly recapitulate the human disease. Given the differences in complexity between human and rodent astrocytes (Oberheim et al., 2009), the contribution of astrocytes to disease progression is most likely under-represented in the animal models. By generating astrocytes from AD patients with mutant PSEN1, we show that the pathogenic PSEN1 DE9 mutation leads to a severe phenotype in AD astrocytes, affecting Ab production, cytokine secretion, Ca 2+ homeostasis, mitochondrial metabolism, ROS production, and lactate secretion, and provides evidence of the importance of astrocytes in AD pathology.
One of the hallmarks of AD pathology is the accumulation of Ab peptides in the patient's brain. Astrocytes are thought to play a critical role in Ab clearance (Ries and Sastre, 2016), while neurons have generally been considered as the main Ab producers (Zhao et al., 1996). However, astrocytes may also contribute to Ab production (Liao et al., 2016;Zhao et al., 2011). Our iPSC-derived AD astrocytes both secrete increased levels of Ab1-42 and show decreased uptake, suggesting that astrocytes contribute to the amyloid plaque formation in AD by both increased release and compromised clearance of Ab1-42. PSEN1 mutations have previously been reported to both activate and inactivate g-secretase activity (Sun et al., 2017;Veugelen et al., 2016;Xia et al., 2015). In our iPSC-derived cells, PSEN1 DE9 mutation had no effect on the overall enzymatic activity of g-secretase and one copy of the PSEN1 DE9 deletion significantly increased Ab1-42 secretion in both astrocytes and neurons, while in a recent report the PSEN1 DE9 point mutation was shown to increase Ab1-40 production in AD neurons (Woodruff et al., 2013). Several factors may contribute to the discrepancies seen between different studies. Vast clinical heterogeneity is seen between different patients with the PSEN1 DE9 mutation (Crook et al., 1998;Hiltunen et al., 2000), suggesting putative variability also in Ab processing, which is likely to be context dependent and cell-type dependent. Furthermore, different studies have used different methods for assessing g-secretase activity. We looked for general enzymatic activity while some studies have examined specific substrates such as N-cadherin (Woodruff et al., 2013), which may further complicate comparison of different studies. iPSC-derived cells may help determine the factors contributing to these controversies in g-secretase activity and Ab production among different studies.
Ca 2+ homeostasis has been proposed to play a crucial role in AD disease progression (Berridge, 2011;. PSEN1 is known to have a direct function in Ca 2+ signaling, and mutations in PSEN1 disturb ER Ca 2+ pools (Bezprozvanny and Mattson, 2008;Ito et al., 1994). However, the mechanisms of how PSEN1 mutations affect Ca 2+ homeostasis are not clear. Increase in expression or activity of intracellular Ca 2+ channels such as RyR or IP 3 R has been proposed (Chan et al., 2000;Cheung et al., 2008), as well as activation of SERCA Ca 2+ pumps . Furthermore, presenilins themselves have been suggested to form passive Ca 2+ leak channels in the ER (Kuo et al., 2015;Tu et al., 2006). In the present study, PSEN1 mutant AD astrocytes showed increased passive Ca 2+ leak from the ER, which could result from the accumulation of full-length PS-1 protein also shown in this study, and its putative ability to form Ca 2+ leak channels in the ER.
Emerging evidence suggests that inflammation actively contributes to AD pathology . Astrocytes are well known to respond to, produce, and secrete many cytokines, and they can contribute to both pro-inflammatory and anti-inflammatory signaling (Sofroniew, 2014). The release of cytokines is known to change during AD disease progression (Heneka et al., 2015), and cytokine levels in the cerebrospinal fluid have been considered as putative biomarkers for AD disease progression. Accordingly, we observed that inflammatory stimulation led to altered cytokine release from AD astrocytes when compared with control cells and, interestingly, g-secretase inhibition was able to partially normalize this, suggesting that the inflammatory response is related to Ab pathology. Thus, iPSC-derived cells may provide a new tool to identify early alterations in cytokine release that could be used as biomarkers for the disease.
Oxidative stress is considered an early event preceding Ab deposits and has been proposed to play a crucial role in AD pathology (Nunomura et al., 2001;Pratico et al., 2001). Neurons are the highly respirative cells in the brain (Belanger et al., 2011), and mitochondrial respiration is a major producer of ROS. We show here that the PSEN1 DE9 mutation switches the metabolism of AD astrocytes toward oxidative phosphorylation, whereas control cells are more glycolytic, as is typical for astrocytes (Belanger et al., 2011). Moreover, treatment with g-secretase inhibitor did not attenuate the

. AD Astrocytes Show Altered Cytokine Release in Pro-inflammatory Conditions
Concentrations of IL-2, IL-6, IL-10, GM-CSF, and CCL5 were quantified from media after stimulation with TNFa (50 ng/mL) and IL-1b (10 ng/mL) for 48 hr with CBA assay. Results are shown as fold change to control lines. DAPT: cells were treated with g-secretase inhibitor DAPT simultaneously with TNFa and IL-1b stimulation. Data are presented as mean ± SEM from three independent experiments (CTRL, n = 6 lines; AD, n = 6 lines; **p < 0.01, ***p < 0.001). See also Figure S3.
changes in mitochondrial metabolism, indicating that this phenotype could be independent of the Ab pathology. The increase in respiratory function leads to increased ROS production by astrocytes, suggesting that astrocytes contribute to increased oxidative stress in AD brain. Furthermore, the concomitant decrease in glycolytic activity resulted in reduced lactate production, thus disturbing the astrocyteneuron lactate shuttling and compromising energy supply Results are normalized to protein content. Three independent experiments (n = 30 replicates/group from 2 isogenic pairs). (B) Extracellular acidification rate following sequential additions of 10 mM glucose (a) and 1 mM oligomycin (b). Results are normalized to protein content. Three independent experiments (n = 30 replicates/group from 2 isogenic pairs). (C-E) Basal respiration (C) and basal glycolysis (D) were quantified after glucose addition from OCR and ECAR curves, respectively. The OCR/ ECAR ratio (E) was calculated after glucose addition to determine the metabolic profile of astrocytes. DAPT: cells were treated with g-secretase inhibitor DAPT before experiments. ***p < 0.001. (F) Representative median fluorescent intensity FACS histograms from CellROX analysis. Non-stained cells are shown in gray, isogenic control cells in black, and AD cells in turquoise. Menadione (MND)-treated cells (violet) were used as a positive control. (G) Quantification of ROS production with CellROX green probe showing median fluorescent intensities (MFI) as a percentage of control group. Three independent experiments (n = 25-30 replicates/group from 2 isogenic pairs; ***p < 0.001). (H) Lactate release was quantified from media with an enzymatic assay and normalized to protein content. Three independent experiments (n = 30 replicates/group from 2 isogenic pairs; ***p < 0.001). All data are presented as mean ± SEM. See also Figure S1.
to neurons (Figley, 2011;Pellerin and Magistretti, 1994). As rat studies have shown that lactate produced and released by astrocytes is essential for memory formation (Suzuki et al., 2011), the reduced lactate secretion by astrocytes might well contribute to dementia in AD.
The importance of astrocytes in neurodegenerative disorders such as AD is becoming more and more evident with accumulating data (Birch, 2014). Recent iPSC-based studies have shown aberrant morphological changes in AD astrocytes (Jones et al., 2017), as well as APOE-related neurotrophic disturbances (Zhao et al., 2017). In our study, AD astrocytes were able to alter Ca 2+ signaling activity of healthy control neurons, further proving the importance of proper astrocyte-neuron interplay in AD.
Currently, there are no effective therapy options for AD. Most clinical trials have focused on either reducing the production or inducing the clearance of Ab, but all have thus far failed (Castello et al., 2014;Golde et al., 2011). However, new approaches are tested constantly, and a recent trial with antibody-based immunotherapy against Ab showed promise (Sevigny et al., 2016). Dysregulated Ca 2+ homeostasis has also been proposed as a putative therapeutic target in AD (Briggs et al., 2017), and a few trials with dantrolene, an RyR inhibitor, have been promising. For example, a shortterm treatment was shown to reduce neuropathology in AD mice (Peng et al., 2012). A third treatment strategy aims at reducing oxidative stress (Gella and Durany, 2009). Our AD astrocytes secrete considerable amounts of Ab1-42, show altered Ca 2+ homeostasis, and produce increased amounts of ROS, thus providing a unique tool for preclinical treatment trials with all these major approaches.
In conclusion, our data show that PSEN1 mutant astrocytes manifest a severe disease phenotype and are likely to contribute significantly to AD progression. Furthermore, as the cells manifest hallmarks of the disease and the major targets for therapeutics, they provide an excellent platform for drug trials.

Generation of iPSCs
Dermal biopsies and blood samples were collected after informed consent and approval from the committee on Research Ethics of Northern Savo Hospital district (license no. 123/2016). Fibroblasts (D) Quantification of the Ca 2+ amplitudes in response to glutamate and glycine application. The x axis shows the genotype of the astrocytes (isogenic CTRL, n = 35 cells; AD, n = 134 cells from 3 independent experiments with 2 isogenic pairs; **p < 0.01). (E) Quantification of the Ca 2+ amplitudes in response to GABA application. The x axis shows the genotype of the astrocytes (isogenic CTRL, n = 27 cells; AD, n = 132 cells from 3 independent experiments with 2 isogenic pairs; ***p < 0.001). Data are presented as mean ± SEM. See also Figure S1. and peripheral blood mononuclear cells T cells were isolated and cultured as previously described (Korhonen et al., 2015;Qu et al., 2013). Somatic cells were reprogrammed to iPSCs with either lentiviral vectors, CytoTune -iPS 1.0, or CytoTune -iPS 2.0 Sendai Reprogramming Kits (Invitrogen) as previously described (Holmqvist et al., 2016). iPSCs were grown on Matrigel-coated (Corning) plates in Essential 8 Medium (E8; Life Technologies) and passaged with 0.5 mM EDTA in the presence of 5 mM Y-27632 ROCK inhibitor (Selleckchem). Isogenic control lines were generated according to a previously published protocol (Chen et al., 2015). Further details are provided in Supplemental Experimental Procedures.

Glucose Uptake Assay
Glucose uptake was determined as previously published (Yamamoto et al., 2015) with slight modifications. In brief, cells were loaded with glucose-free medium in the absence or presence of 120 mM 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG, Thermo Fisher) for 30 min in 5% CO 2 at 37 C. The 2-NBDG uptake was stopped by washing the cells twice with PBS. Cells were detached with Accutase and resuspended in PBS prior to flow-cytometric measurement. Data from 15,000 single live cell events were collected using FACSCalibur (BD Biosciences, San Jose, CA, USA).

Glutathione Secretion Assay
Glutathione measurements were performed as described earlier (Liddell et al., 2006) by mixing the medium with an equal volume of cold sulfosalicylic acid (1% in water). Samples were further diluted 1:5 with water before the measurements. Glutathione con-centrations were determined from standard curve and normalized to total protein amount using a Pierce BCA protein assay according to the manufacturer's instructions (Thermo Fisher).

Ab and g-Secretase Assays
Ab1-40 and Ab1-42 amounts were measured from the culture medium conditioned for 72 hr with ELISA according to the manufacturer's instructions (Invitrogen). DAPT (5 mM; Sigma) was added to the medium 1 day prior to the conditioning when applicable. Results were normalized to total protein concentration (Pierce BCA protein assay). Fluorochrome-conjugated Ab1-42 (HiLyte Fluor 488-labeled, AnaSpec) was mixed with non-fluorescent fibrilized Ab1-42 (from American Peptides, incubated at +37 C for 1 week) in a 1:15 ratio and added to the cells at a final concentration of 5 mM. Cells were incubated overnight, washed three times with PBS to remove non-internalized Ab, and detached with Accutase. The percentage of 488-positive cells was determined with FACSCalibur (BD Biosciences). g-Secretase activity was measured following a previously published protocol (Farmery et al., 2003;Viswanathan et al., 2011). g-Secretase inhibitor L685,458 (Sigma) was included for assay validation purposes.

Ca 2+ Imaging
Ca 2+ signals from astrocytes were recorded with a confocal microscope, and data were analyzed as previously described (Mutikainen et al., 2016). All experiments were carried out at 37 C. Cells were loaded with Fluo4 (Fluo-4-acetoxymethyl [AM]-ester, Life Technologies; 5 mM for 20 min), rinsed with Hank's balanced salt solution (HBSS) containing 2 mM CaCl 2 , and transferred to the recording chamber (Cell MicroControls; flow rate approximately 1-2 mL/min, chamber volume 0.4 mL). Ca-free 1 mM EGTA-containing HBSS was applied for 3 min for baseline recordings. For the estimation of non-specific Ca 2+ leak from the ER, 1 mM thapsigargin, 50 mM ryanodine, and 100 mM 2APB were applied. All compounds were diluted in Ca 2+ -free HBSS with 1 mM EGTA. To investigate intercellular propagation of Ca 2+ waves, we used local electrical stimulation. A glass patch pipette (1 MU) filled with bath solution was placed close to an astrocyte. A platinum stimulating electrode was placed into the pipette and an additional two ground platinum electrodes were placed in the bath area at both sides of the cell. Two-second square stimulation trains at 20-Hz frequency with pulse duration of 2 ms were used.
For co-culture experiments, cells were first loaded with Fluo4, followed by a 10-min washout with a basic solution containing 152 mM NaCl, 10 mM glucose, 10 mM HEPES, 2.5 mM KCl, 2 mM CaCl 2 , and 1 mM MgCl 2 (pH 7.4). Cells were then transferred to the recording chamber continuously rinsed with the same basic solution. Fluorescent signals were acquired with an Olympus IX70 microscope on the Till Photonics imaging setup (FEI) equipped with a 12-bit CCD Camera (SensiCam) with a light excitation wavelength of 494 nm and adequate filters. Glutamate (100 mM; with the co-agonist glycine 10 mM) or GABA (100 mM) were applied for 2 s by a fast perfusion system . Depolarizing solution containing 30 mM KCl was then applied to distinguish neurons from other cell types. At the end, cells were challenged with ionomycin (10 mM, 2 s) for normalization of the tested signals.

Cytometric Bead Array
A bead-based multiplex assay was used to analyze the cytokine secretion. Cytokine concentrations were detected by the cytometric bead array (CBA) Flex Sets (BD Biosciences) Human Soluble Protein (CCL5/RANTES, GM-CSF, and IL-6) and Human Enhanced Sensitivity (IL-2 and IL-10). The assay was done according to the manufacturer's instructions with minor modifications. For Soluble Protein, 20 mL of sample or cytokine standard and 20 mL of 1:50 bead mixture was used and for Enhanced Sensitivity, 25 mL of sample or cytokine standard and 10 mL of 1:20 bead mixture was used. Beads were acquired on FACSAriaIII (BD Biosciences). At least 300 events per cytokine were measured. Data were analyzed using FCAP Array 2.0 (SoftFlow, Hungary) and cytokine concentrations were calculated by regression analysis from known standard concentrations.

Cellular Metabolism, ROS, and Lactate Analysis
Cellular metabolism was analyzed with the Seahorse XF24 analyzer and Mito Stress Test according to the manufacturer's instructions (Agilent Technologies). Assay medium was supplemented with 13 Glutamax and 0.5 mM sodium pyruvate. Glucose (10 mM) was added during the assay. Final concentrations of oligomycin, FCCP (carbonyl cyanide-4-phenylhydrazone), antimycin, and rotenone were 1 mM each. Results were normalized to total protein content (Pierce BCA protein assay). For ROS measurement, cells were loaded with 1 mM CellROX green reagent (Molecular Probes) for 45 min. Menadione (50 mM; Sigma) was used as a positive control and added together with CellROX. Median fluorescent intensities (MFI) of 10,000 single cell events from each sample were collected using FACSCalibur (BD Biosciences). L(+)-lactate was measured from the conditioned medium (24 hr) using a colorimetric Lactate Assay kit (Sigma) according to the manufacturer's instructions. Results were normalized to total protein amount (Pierce BCA protein assay).

3D Co-cultures
Thin-layer Matrigel co-cultures were prepared as described in Choi et al. (2014) with slight modifications. In brief, equal amounts of iPSC-derived astrocytes and neurons were mixed and resuspended with 1:10 diluted Matrigel to a final concentration of 1 3 10 6 cells/mL. Cell suspension was plated on poly-L-ornithinecoated coverslips and left to polymerize overnight. Wells were filled with NDM medium on the next day and the medium was changed twice a week. Cells were kept in co-cultures for 4-6 weeks before experiments.

Statistical Analyses
Statistical analyses were performed with GraphPad Prism 5.03 software (GraphPad Software) using Student's t test or one-way ANOVA with Tukey's post hoc test. For calcium imaging, data were analyzed with Origin9 software (OriginLab, Northampton, MA, USA) using one-way ANOVA with Fisher's post hoc test or Student's t test. Statistical significance was assumed at p < 0.05. All data are expressed as mean ± SEM.