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

Highlights

• PSEN1 mutant AD astrocytes manifest hallmarks of AD pathology

• Altered mitochondrial metabolism in AD astrocytes increases oxidative stress

• AD astrocytes reduce the calcium signaling activity of healthy neurons

• Astrocytes are important in the pathogenesis of AD

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 β-amyloid (Aβ) 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 Aβ 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 tumor-derived 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 Ca²⁺ 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 ΔE9) (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 ΔE9 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 ɛ3/ɛ3 isoforms of APOE, the most important risk gene for LOAD. To examine the cause-effect relationship between ΔE9 mutation and AD phenotype, we also generated gene-corrected isogenic control lines from one symptomatic AD patient and the pre-symptomatic PSEN1 ΔE9 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).

Table 1Summary of the Healthy Controls and Patients Used in This Study

Patient	Sex	Age When Sample Taken (Years)	PSEN1Genotype	APOEGenotype	Status	Sample Type	Isogenic Control Line
Ctrl1	F	adult	normal	ɛ3/ɛ3	normal	skin biopsy	–
Ctrl2	M	62	normal	ɛ3/ɛ3	normal	skin biopsy	–
Ctrl3	F	44	normal	ɛ3/ɛ3	normal	skin biopsy	–
AD1	F	64	ΔE9	ɛ3/ɛ3	Alzheimer’s disease	blood sample	–
AD2	M	48	ΔE9	ɛ3/ɛ3	Alzheimer’s disease	skin biopsy	PSEN1ΔE9 corrected
AD3	F	47	ΔE9	ɛ3/ɛ3	pre-symptomatic	skin biopsy	PSEN1ΔE9 corrected

See also Table S1; Figures S1 and S2.

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 S100β 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 Ca²⁺ waves (Figures 1G–1I and S3A). No evident differences were seen in the differentiation potential between the genotypes, and both AD and control iPSCs generated comparable, functional astrocytes.

PSEN1 ΔE9 Mutant Astrocytes Contribute to β-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 γ-secretase complex. Since PSEN1 ΔE9 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 wild-type PS-1 (Thinakaran et al., 1996), we analyzed PS-1 endoproteolytic cleavage. As expected, PSEN1 ΔE9 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 γ-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 Aβ production, but astrocytes are also able to secrete Aβ (Liao et al., 2016). Thus, we quantified Aβ1–40 and Aβ1–42 from the astrocyte culture media. The secretion of Aβ1–42 was increased about 5-fold in the AD cultures when compared with the controls, whereas Aβ1–40 secretion was not altered (Figures 2D, 2E, S4A, and S4B). This led to a significantly increased Aβ1–42/Aβ1–40 ratio and toxic Aβ profile in AD astrocytes (Figures 2F and S4C). Treatment with the γ-secretase inhibitor DAPT decreased the production of Aβ1–40 and completely blocked the production of Aβ1–42 in both AD and control astrocytes (Figures 2D and 2E). The overall Aβ 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 Aβ-producing cells. Importantly, both the PS-1 endoproteolytic processing and Aβ 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 Aβ1–42 by fluorescence-activated cell sorting (FACS). After 16 hr of incubation with a fluorochrome-conjugated fibrilized Aβ1–42, internalized Aβ was quantified and AD astrocytes showed reduced capacity to take up Aβ when compared with controls (Figure 2H). These results show that PSEN1 ΔE9 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 Aβ1–42.

Ca²⁺ Signaling in the ER Is Disturbed in PSEN1 ΔE9 Mutant Astrocytes

Several PSEN1 mutations, including PSEN1 ΔE9, have been reported to disturb Ca²⁺ release from the ER (Cedazo-Minguez et al., 2002, Ito et al., 1994). Thus, we next analyzed ER Ca²⁺ cycling and especially Ca²⁺ leakage from the ER. The non-specific Ca²⁺ leakage was studied by simultaneous blocking of ryanodine receptor (RyR), inositol triphosphate receptor (IP₃R), and SERCA (Figure 3A). The rate of the non-specific Ca²⁺ release, represented by the slope of the increase in [Ca²⁺]_i level, was faster in AD than in control astrocytes (Figures 3B and S5). These results show that PSEN1 ΔE9 AD astrocytes manifest altered cellular Ca²⁺ homeostasis.

Cytokine Secretion after Inflammatory Stimulation Is Altered in PSEN1 ΔE9 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-1β (IL-1β) (10 ng/mL) and tumor necrosis factor α (TNFα) (50 ng/mL). Stimulation of control astrocytes with IL-1β and/or TNFα 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-1β and TNFα and compared the cytokine secretion between AD and control cells. Upon inflammatory stimulation, PSEN1 ΔE9 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 ΔE9 cultures (Figure 4). Interestingly, treatment with γ-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 ΔE9 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 ΔE9 astrocytes were more oxidative than isogenic control cells, which relied more on glycolysis as typical for astrocytes (Figures 5C–5E). Treatment with the γ-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 ΔE9 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 ΔE9 astrocytes (Figure 5H). These data show that PSEN1 ΔE9 astrocytes are more oxidative than the glycolytic control astrocytes, generating more ROS and oxidative stress, and producing less lactate.

PSEN1 ΔE9 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 ΔE9 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 ΔE9 mutant astrocytes (Figure 6A). Application of glutamate, together with the co-agonist glycine, resulted in significantly lower Ca²⁺-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 Ca²⁺ transients evoked by γ-aminobutyric acid (GABA) (Figures 6B, 6C, and 6E). These results show that the PSEN1 ΔE9 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 ΔE9 mutation leads to a severe phenotype in AD astrocytes, affecting Aβ production, cytokine secretion, Ca²⁺ 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 Aβ peptides in the patient’s brain. Astrocytes are thought to play a critical role in Aβ clearance (Ries and Sastre, 2016), while neurons have generally been considered as the main Aβ producers (Zhao et al., 1996). However, astrocytes may also contribute to Aβ production (Liao et al., 2016, Zhao et al., 2011). Our iPSC-derived AD astrocytes both secrete increased levels of Aβ1–42 and show decreased uptake, suggesting that astrocytes contribute to the amyloid plaque formation in AD by both increased release and compromised clearance of Aβ1–42. PSEN1 mutations have previously been reported to both activate and inactivate γ-secretase activity (Sun et al., 2017, Veugelen et al., 2016, Xia et al., 2015). In our iPSC-derived cells, PSEN1 ΔE9mutation had no effect on the overall enzymatic activity of γ-secretase and one copy of the PSEN1 ΔE9 deletion significantly increased Aβ1–42 secretion in both astrocytes and neurons, while in a recent report the PSEN1 ΔE9 point mutation was shown to increase Aβ1–40 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 ΔE9mutation (Crook et al., 1998, Hiltunen et al., 2000), suggesting putative variability also in Aβ processing, which is likely to be context dependent and cell-type dependent. Furthermore, different studies have used different methods for assessing γ-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 γ-secretase activity and Aβ production among different studies.

Ca²⁺ homeostasis has been proposed to play a crucial role in AD disease progression (Berridge, 2011, Green and LaFerla, 2008). PSEN1 is known to have a direct function in Ca²⁺ signaling, and mutations in PSEN1 disturb ER Ca²⁺ pools (Bezprozvanny and Mattson, 2008, Ito et al., 1994). However, the mechanisms of how PSEN1 mutations affect Ca²⁺ homeostasis are not clear. Increase in expression or activity of intracellular Ca²⁺ channels such as RyR or IP₃R has been proposed (Chan et al., 2000, Cheung et al., 2008), as well as activation of SERCA Ca²⁺pumps (Green et al., 2008). Furthermore, presenilins themselves have been suggested to form passive Ca²⁺ leak channels in the ER (Kuo et al., 2015, Tu et al., 2006). In the present study, PSEN1 mutant AD astrocytes showed increased passive Ca²⁺ 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 Ca²⁺ leak channels in the ER.

Emerging evidence suggests that inflammation actively contributes to AD pathology (Zhang et al., 2013). 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, γ-secretase inhibition was able to partially normalize this, suggesting that the inflammatory response is related to Aβ 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 Aβ 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 ΔE9 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 γ-secretase inhibitor did not attenuate the changes in mitochondrial metabolism, indicating that this phenotype could be independent of the Aβ 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 astrocyte-neuron lactate shuttling and compromising energy supply 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 Ca²⁺ 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 Aβ, 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 Aβ showed promise (Sevigny et al., 2016). Dysregulated Ca²⁺ 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 short-term 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 Aβ1–42, show altered Ca²⁺ homeostasis, and produce increased amounts of ROS, thus providing a unique tool for pre-clinical 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.