Abstract
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the progressive death of motor neurons (MNs). MN degeneration in ALS involves both cell-autonomous and non-cell autonomous mechanisms, with glial cells playing important roles in the latter. More specifically, astrocytes with mutations in the ALS-associated gene Cu/Zn superoxide dismutase 1 (SOD1) promote MN death. The mechanisms by which SOD1-mutated astrocytes reduce MN survival are incompletely understood. In order to characterize the impact of SOD1 mutations on astrocyte physiology, we generated astrocytes from human induced pluripotent stem cell (iPSC) derived from ALS patients carrying SOD1 mutations, together with control isogenic iPSCs. We report that astrocytes harbouring SOD1(A4V) and SOD1(D90A) mutations exhibit molecular and morphological changes indicative of reactive astrogliosis when compared to matching isogenic astrocytes. We show further that a number of nuclear phenotypes precede, or coincide with, reactive transformation. These include increased nuclear oxidative stress and DNA damage, and accumulation of the SOD1 protein in the nucleus. These findings reveal early cell-autonomous phenotypes in SOD1-mutated astrocytes that may contribute to the acquisition of a reactive phenotype involved in alterations of astrocyte-MN communication in ALS.
1. Introduction
Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron (MN) disease characterized by the progressive degeneration of MNs in the cerebral cortex, brain stem and spinal cord, resulting in gradual muscle paralysis and ultimately death by respiratory failure (Mejzini et al., 2019; Kim et al., 2020). The majority of ALS cases are sporadic, with less than 20% of cases inherited through families (familial ALS). Multiple deleterious variants in numerous genes have been associated with familial ALS over the years, starting with the discovery of the first genetic mutation to cause ALS, affecting the Cu/Zn superoxide dismutase 1 (SOD1) gene (Rosen et al., 1993). Together with SOD1, chromosome 9 open reading frame 72 (C9orf72), TAR DNA binding protein (TARDBP), and FUS RNA binding protein (FUS) are the most frequently mutated genes in ALS, accounting for approximately 70% of familial ALS cases (Mejzini et al., 2019; Kim et al., 2020; Brenner and Freischmidt, 2022). Although these genes play multiple functions in disease pathogenesis, many of which remain to be fully elucidated, protein misfolding and accumulation of toxic aggregates is a common feature of the most common familial ALS mutations (Calabrese et al., 2022; Tran and Lee, 2022; Arnold et al., 2023). Perturbations of numerous mechanisms contribute to MN death in ALS, including, but not limited to, oxidative stress, RNA metabolism and protein homeostasis, nucleocytoplasmic trafficking, dynamics of ribonucleoprotein bodies, mitochondrial functions, and autophagy (Balendra and Isaacs, 2018; Burk and Pasterkamp, 2019; Mejzini et al., 2019; Prasad et al., 2019).
Although traditionally defined as a disease that affects vulnerable MNs, there is increasing evidence that the convergence of damage within multiple cell types is crucial to MN loss in ALS. Similar to other neurodegenerative diseases, ALS is characterized by extensive neuroinflammation involving astrogliosis, activation of microglia, and infiltration of peripheral immune cells at sites of neuronal degeneration (Beers and Appel, 2019; Cipollina et al., 2020). Reactive astrogliosis is a hallmark of post-mortem tissue from ALS patients and ALS mouse models (Schiffer et al., 1996; Hall et al., 1998; Shibata et al., 2001; Johann et al., 2015). Several studies with cultured cells provide evidence suggesting that astrocytes harbouring ALS mutations can be toxic to MNs in vitro (Di Giorgio et al., 2007; Nagai et al., 2007; Haidet-Phillips et al., 2011). The contribution of astrocytes to MN pathology in ALS is complex, depending on the stage of disease progression. It is hypothesized that astrocyte reactive transformation initially occurs as a neuroprotective response during early stages of ALS. At least some activated astrocytes can then gradually become neuroinflammatory during disease progression, contributing to neuronal degeneration. The deleterious effects of astrocytes on MNs in ALS may result from loss of supportive functions and/or gain of toxic activities, such as secretion of neuroinflammatory molecules (Meyer et al., 2014; Varcianna et al., 2019; Guttenplan et al., 2020; Van Harten et al., 2021; Arredondo et al., 2022).
Approximately 15% of familial ALS case are caused by mutations in the SOD1 gene, which encodes an abundant and broadly expressed protein that catalyzes dismutation of superoxide to hydrogen peroxide and molecular oxygen thereby protecting cells from reactive oxygen species toxicity. SOD1 is present in the cytosol, mitochondria, peroxisomes and nuclei (Okado-Matsumoto and Fridovich, 2001; Bunton-Stasyshyn et al., 2015; Xu et al., 2022). A large number of genetic variants of SOD1 have been identified in ALS patients. SOD1 mutations usually result in gain-of-function effects in which the mutated SOD1 proteins acquire new toxic functions thought to derive from misfolding and an increased predisposition to aggregation. Although the pathogenic mechanisms of aggregated SOD1 in MNs remain to be fully elucidated, they affect key cellular processes such as scavenging of free radicals, mitochondrial function, axonal transport, protein quality control, and mRNA splicing, to name a few (Bunton-Stasyshyn et al., 2015; Abati et al., 2020; Kim et al., 2020; Peggion et al. 2022).
In addition to affecting MN physiology, SOD1 mutations have an impact on astrocyte biology and the cross-talk between astrocytes and MNs. More specifically, astrocytes harbouring SOD1 mutations decrease MN survival both in vivo and in vitro (Di Giorgio et al., 2007; Nagai et al., 2007; Marchetto et al., 2008; Meyer et al., 2014). This effect is mediated by cell-to-cell signaling between astrocytes and MNs (Nagai et al., 2007; Fritz et al., 2013; Urban et al., 2023). Little information is available on how SOD1 mutations drive intrinsic changes within astrocytes that affect their cross-talk with MNs. We report that the SOD1(A4V) and SOD1(D90A) mutations are associated with enhanced astrocyte reactivity in the absence of other cell types. Reactive astrogliosis is concomitant with increased DNA damage and accumulation of SOD1 in the nucleus. These changes are preceded by signs of increased nuclear oxidative stress. These findings reveal early nuclear phenotypes in SOD1-mutated astrocytes that may contribute to the acquisition of a reactive phenotype involved in mechanisms of MN degeneration in ALS.
2. Results
2.1. Generation of ventral spinal cord-like astrocytes from SOD1-mutated human iPSCs
Induced cells with molecular features of ventral spinal cord astrocytes were generated from human iPSC lines derived from ALS patients harbouring SOD1(A4V) or SOD1(D90A) mutations as described (Soubannier et al., 2022). The A4V mutation is the most frequent SOD1 mutation in North America, while the D90A mutation is the most prevalent in Europe (Kim et al., 2020). Matching gene-edited iPSC lines were used as controls (hereafter termed Iso(A4V) and Iso(D90A) to indicate which specific mutations were corrected). Immunocytochemistry and RT-PCR confirmed the initial generation of neural progenitor cells (NPCs) with caudal and ventral neural tube properties, such as expression of the cervical marker HOXA5 and ventral marker NKX6.1 (Supplementary Figure 1 – Fig. S1). Upon exposure to pro-astrogenic culture conditions, these validated NPCs generated robust numbers of cells exhibiting a fibrous morphology and the co-expression of typical astrocyte markers, such as GFAP and S100B, as early as 30 days after the start of in vitro differentiation (DIV30). Comparable yields of GFAP+/S100B+ cells were observed when SOD1-mutated NPCs or their isogenic counterparts were used (Fig. 1A-D). Exposure of Iso(A4V) astrocytes to a combination of tumour necrosis factor-alpha (TNF-α), interleukin 1-alpha (IL-1α), and complement component 1, subcomponent q (C1q), previously shown to promote astrocyte reactivation (Liddelow et al., 2017), resulted in upregulation of several astrogliosis marker genes, such as complement component 1, subcomponent s (C1s), complement component 3 (C3), S100 calcium binding protein A10 (S100A10), pentraxin 3 (PTX3), serpin family g member 1 (SERPING1), c-c motif chemokine ligand 2 (CCL2), and C-X-c motif chemokine ligand 10 (CXCL10) (Fig. 1E). Consistently, a number of cytokines and chemokines were over-secreted by Iso(A4V) astrocytes treated with TNF-α, IL-1α, and C1q, compared to untreated cells (Fig. 1F). These observations show that iPSC-derived astrocytes display properties similar to physiological astrocytes.
A-D) Representative images of phase contrast (left-hand panel in each row) and double-labeling immunofluorescence of GFAP (green) and S100B (red) expression in DIV60 astrocytes generated from iPSC lines with A4V or D90A mutations in SOD1, and their matching isogenic lines (termed Iso(A4V) and Iso(D90A) to indicate which mutations were corrected); Hoechst counterstaining (blue) is shown. The majority of induced cells express S100B, and a significant proportion of S100B-positive cells co-express GFAP at high level, while other S100B-positive cells co-express GFAP at lower levels. E) Real-time PCR analysis of the expression of the indicated reactive astrocyte markers in DIV60 Iso(A4V) astrocytes treated, or not, with IL-1α, TNF-α, and C1q. (F) List of the ten most increased cytokines and chemokines secreted in the medium by Iso(A4V) astrocytes following treatment with TNF-α, IL-1α, and C1q compared to treatment with vehicle. Differences are expressed as fold change.
2.2. SOD1-mutated astrocytes undergo reactive transformation
Previous studies have shown increased reactive transformation in astrocytes harbouring mutations in different familial ALS genes, including SOD1, C9orf72, FUS, and valosin-containing protein (VCP) (Birger et al., 2019; Taha et al., 2022; Stoklund Dittlau et al., 2023). Much remains to be learned about the cellular mechanisms underlying reactive astrogliosis in ALS, particularly cell-autonomous processes. In this context, we tested whether cultures of astrocytes harbouring the SOD1(A4V) mutation would undergo reactive transformation in the absence of extrinsic cues. Morphological comparison of SOD1(A4V) astrocytes and their isogenic counterparts using phalloidin staining to label cytoskeletal structures revealed no significant differences at DIV30 (Fig. 2A). Since reactive astrogliosis is usually characterized by a disassembly of F-actin stress fibers stained with phalloidin into a more disorganized G-actin network (Hansson, 2015; Tyzack et al., 2017), this observation suggested the lack of significant reactivation in SOD1(A4V) astrocytes at DIV30. To test this possibility further, we conducted quantitative RT-PCR studies to compare the expression levels of genes known to be up-regulated in reactive astrocytes (Liddelow et al., 2017). These studies showed a trend toward increased levels of C1s and S100A10 in SOD1(A4V) astrocytes, but no significant difference in the expression of other reactive astrocyte phenotype markers, such as C3, PTX3, and SERPING1 (Fig. 2B). These findings suggest that SOD1(A4V) astrocytes are not significantly more reactive than isogenic astrocytes after 30 days in vitro.
A, B) DIV30 astrocytes. A) Representative images of the actin cytoskeleton of SOD1(A4V) and matching isogenic astrocytes visualized by Alexa488-conjugated phalloidin staining (green); Hoechst counterstaining (blue) is shown. B) Real-time PCR analysis of the expression of the indicated reactive astrocyte markers in SOD1(A4V) and matching isogenic astrocytes. C, D) DIV60 astrocytes. C) Representative images of the actin cytoskeleton of SOD1(A4V) and matching isogenic astrocytes visualized by phalloidin staining (green); Hoechst counterstaining (blue) is shown. D) Real-time PCR analysis of the expression of the indicated reactive astrocyte markers in either SOD1(A4V) and isogenic astrocytes (top row) or SOD1(D90A) and isogenic astrocytes (bottom row). Statistical analyses were performed with Student’s t-test, graphs show mean ± SEM; *p < 0.05; ****p < 0.0001; n = 3. E) List of the ten most increased cytokines and chemokines secreted in the medium by SOD1(A4V) astrocytes compared to Iso(A4V) astrocytes. Differences are expressed as fold change.
We next performed the same studies at DIV60, when induced astrocytes are more developmentally mature. Phalloidin staining showed that isogenic astrocytes continued to exhibit the presence of F-actin stress fibers typical of healthy cells, whereas SOD1(A4V) astrocytes displayed a disassembly of the stress fibers and the presence of actin networks characterized by ring-like structures, ruffles, and radial actin filaments, suggestive of reactive transformation (Fig. 2C). In agreement with this observation, C1s, C3, S100A10, SERPING1, and PTX3 were all upregulated in SOD1(A4V)-mutated astrocytes at DIV60 (Fig. 2D). Moreover, SOD1(A4V) astrocytes exhibited increased secretion of several proteins associated with a reactive phenotype (Fig. 2E). Similar morphological and gene expression phenotypes were observed at DIV60 in astrocytes harbouring the SOD1(D90A) mutation, showing that these phenotypes were not unique to the A4V mutation in SOD1 (Fig. 2C, D; Fig. S2). Together, these results provide evidence that astrocytes with SOD1(A4V) and SOD1(D90A) mutations undergo cell-autonomous reactive transformation by 60 days in vitro when compared to their isogenic counterparts.
2.3. Increased nuclear oxidative stress in SOD1-mutated astrocytes
To further characterize the phenotype of SOD1-mutated astrocytes, we first tested whether we could detect differences with isogenic astrocytes preceding detectable signs of reactive transformation. Based on previous studies showing increased oxidative stress in astrocytes carrying ALS mutations (Shibata et al., 2001; Birger et al., 2019; Appel et al., 2021), SOD1-mutated astrocyte cultures were tested for oxidative stress at DIV30 using a range of concentrations of the probe MitoSox, a dye readily oxidized by superoxide ions. In agreement with previous studies, we detected higher MitoSox levels in SOD1(A4V) astrocytes at a concentration of 1 μM (Fig. S3): this experimental condition is expected to detect mainly mitochondrial superoxide, a type of reactive oxygen species (Roelofs et al., 2015). Increasing the concentration of MitoSox to 5 μM, a dose at which this probe redistributes to the nucleus (Roelofs et al., 2015), revealed statistically significantly higher levels of nuclear MitoSox intensities in both SOD1(A4V) and SOD1(D90A) astrocytes, compared to the corresponding isogenic astrocytes (Fig. 3A, B). This finding suggests that astrocytes harbouring these ALS-associated SOD1 mutations have increased generation, or impaired clearance, of superoxide ions within the nucleus as early as 30 days after the start of in vitro differentiation.
A, B) Representative images of nuclear MitoSox (5 μM) fluorescence in either SOD1(A4V) and isogenic astrocytes (A) or SOD1(D90A) and isogenic astrocytes (B). Graphs depict quantifications of nuclear MitoSox intensities in SOD1- mutated astrocytes compared to matching isogenic astrocytes. Statistical analyses were performed with Student’s t-test, graphs show mean ± SEM; ***p < 0.0005; ****p< 0.0001; n = 3 (more than 5,000 cells counted).
2.4. Increased DNA damage and nuclear accumulation of SOD1 protein in SOD1-mutated astrocytes
Considering the established link between oxidative stress and DNA damage in ALS (Kok et al., 2021; Szebényi et al., 2021), we next sought to determine whether the increased nuclear oxidative stress observed in SOD1-mutated astrocytes was correlated with increased DNA damage. To this end, we compared the levels of γH2AX, which represents the phosphorylated form of the histone variant H2AX and is a marker for DNA double strand breaks, in SOD1(A4V) and SOD1(D90A) astrocytes, compared to their isogenic counterparts. These studies revealed no detectable differences at DIV30 (not shown), but by DIV60 we observed an increased γH2AX signal in the nuclei of SOD1(A4V) and SOD1(D90A) astrocytes, indicative of increased double strand DNA break (Fig. 4A, B).
A) Representative images of double-labeling immunofluorescence analysis of ψH2AX and SOD1 in DIV60 SOD1(A4V) and SOD1(D90A) astrocytes together with their matching isogenic controls. B-D) Quantification of nuclear ψH2AX (B), nuclear SOD1 (C), and nucleus vs cytosol SOD1 ratio (D) in DIV60 astrocytes of all four genotypes under study. Statistical analyses were performed with Student’s t-test, graphs show mean ± SEM; *p< 0.05; ***p < 0.0005; ****p< 0.0001; n = 3 (more than 5,000 cells counted).
Previous studies have shown that SOD1 accumulates in the nucleus in response to increased levels of reactive oxygen species and DNA damage (Inoue et al., 2010; Tsang et al., 2014). Moreover, an increase in nuclear versus cytosolic SOD1 protein localization was observed in ALS and other neurodegenerative disorders (Gertz et al., 2012; Bordoni et al., 2019). In the nucleus, in addition to its superoxide dismutase function, SOD1 acts a transcription factor that regulates the expression of oxidative resistance and DNA repair genes (Inoue et al., 2010; Tsang et al., 2014). Thus, we examined the intracellular localization of the SOD1 protein in mutated and isogenic human iPSC-derived astrocytes, both at earlier (DIV30) and later (DIV60) stages of in vitro differentiation. We observed no notable difference in nuclear SOD1 localization in SOD1(A4V) and SOD1(D90A) astrocytes compared to their isogenic counterparts at DIV30 (not shown). In contrast, a significant increase in nuclear SOD1, as detected through quantification of SOD1 signal within the nucleus, was observed in both SOD1(A4V) and SOD1(D90A) astrocytes at DIV60 (Fig. 4C). This observation was supported by quantification of the nucleus/cytosol SOD1 ratio in SOD1-mutated and isogenic astrocytes (Fig. 4D).
Taken together, these results suggest that astrocytes carrying ALS-associated SOD1 mutations undergo early nuclear oxidative stress, which is then correlated with increased DNA damage, nuclear accumulation of SOD1, and reactive transformation at later in vitro stages.
3. Discussion
In this study, we sought to investigate the involvement of astrocytes in ALS, with specific focus on astrocytes harbouring SOD1 mutations. Although rodent models have provided important insight into the contribution of astrocytes to ALS pathophysiology, there is growing evidence that human and murine astrocytes differ at various levels, including morphology, function, and expression of genes enriched in disease-associated pathways (Zhang et al., 2016; Kelley et al., 2018; Hodge et al., 2019). Moreover, most animal models are based on overexpression paradigms that do not fully recapitulate the pathophysiological expression levels occurring in human patients. The use of astrocytes generated from iPSCs derived from ALS patients carrying mutations in the SOD1 gene can address both of these limitations, while also allowing the study of the impact of SOD1 mutations on astrocyte biology in the absence of other cell type like MNs and microglia.
Using human astrocytes generated from iPSCs derived from familial ALS patients with SOD1(A4V) and SOD1(D90A) mutations we observed that these cells acquire a reactive phenotype during the first 60 days of in vitro culture. This cell-autonomous transformation agrees with previous studies showing increased reactivation of astrocytes harbouring mutations in several familial ALS genes (Birger et al., 2019; Taha et al., 2022; Stoklund Dittlau et al., 2023). Under the experimental conditions used in our investigations, SOD1-mutated astrocytes exhibited a “mixed” gene expression profile characterized by upregulation of both neurotoxic (A1 subtype) markers, such as C1s, C3, SERPING1, CXCL10, and neuroprotective (A2 subtype) markers, such as PTX3, S100A10, CCL2 (Liddelow et al., 2017). This finding agrees with previous studies showing up-regulation of both A1 and A2 marker genes in SOD1(D90A)-mutated astrocytes (Taha et al., 2022). These observations suggest that, in the absence of MNs and other cells implicated in ALS pathophysiology, such as microglia, astrocytes with mutations in SOD1 are reactive but not neuroinflammatory under in vitro culture conditions.
In an effort to improve our understanding of the molecular mechanisms underlying the cell-autonomous reactive transformation of SOD1-mutated astrocytes, we observed that oxidative stress is detectable in the nuclei of these cells before overt signs of astrogliosis. To our knowledge, this is the first observation of increased levels of reactive oxygen species in the nucleus of astrocytes harbouring ALS mutations, and one of the earliest cell-autonomous phenotypes detected in these cells thus far. This nuclear phenotype was correlated with increased DNA damage, as well as nuclear accumulation of the SOD1 protein, at later in vitro stages.
These findings are consistent with previous evidence in multiple cell types that SOD1 becomes increasingly localized in the nucleus in response to both oxidative stress and DNA damage (Inoue et al., 2010; Tsang et al., 2014). The observation of nuclear oxidative stress before detection of DNA damage and SOD1 accumulation in the nucleus suggests a temporal sequence of nuclear phenotypes triggered by early nuclear oxidative damage. It remains to be determined whether it is the latter, or the ensuing DNA damage (or both), that contributes to SOD1 nuclear accumulation. SOD1 was previously detected in the nuclei of ventral horn astrocytes in post-mortem samples from ALS patients carrying SOD1 mutations (Forsberg et al., 2011). Those studies could not determine whether this phenotype was dependent on the presence of MNs or other glial cells. Based on our findings, we propose that SOD1 nuclear accumulation in astrocytes harbouring ALS-associated SOD1 mutations is a previously-unrecognized cell-autonomous mechanism. It is reasonable to assume that the increased presence of wild-type SOD1 in the nucleus would normally provide an early antioxidant and/or DNA repair function beneficial to astrocytes. The presence of mutated SOD1, however, would interfere with the physiological function of wild-type SOD1, leaving the cells more vulnerable to oxidative stress and DNA damage.
The nuclear phenotypes discussed above could be contributing factors to the increased reactivity of SOD1-mutated astrocytes compared to their isogenic counterparts. Oxidative stress in ALS SOD1-mutated astrocytes is involved in the neurotoxic effects of these cells on MN, as suggested by the observation that enhanced resistance to oxidative stress through increased levels of either total NAD content or SIRT6 protein can abrogate astrocyte toxicity toward co-cultured MNs (Harlan et al., 2019). This finding is consistent with results showing that astrocytes derived from iPSCs from ALS patients with mutated C9orf72 exhibit increased oxidative stress and neurotoxicity (Birger et al., 2019). Moreover, analysis of post-mortem tissue from sporadic ALS patients revealed that poly(ADP-ribose) polymerase, a key DNA repair protein, is increased in astrocytes, suggesting increased DNA damage in astrocytes in ALS (Kim et al., 2003). Consistently, DNA damage response pathways are affected in both astroglia and neurons in brain organoid slice cultures derived from iPSCs with mutated C9orf72 (Szebényi et al., 2021).
Additionally, evidence that DNA damage can contribute to astrocyte dysfunction in neurodegenerative diseases has recently emerged from the study of astrocytes from Huntington’s Disease patients (Lange et al., 2023). Perturbation of nuclear SOD1 functions is also expected to contribute to enhanced astrocyte reactivation. SOD1 localizes to the nucleus under normal and pathological conditions to contribute to oxidative stress response and DNA repair mechanisms (Inoue et al., 2010; Tsang et al., 2014; Xu et al., 2022). It is therefore reasonable to hypothesize that a dominant-negative effect of mutated SOD1 on the nuclear roles of wild-type SOD1 would lead to increased oxidative stress and DNA damage in astrocytes, thereby contributing to mechanisms promoting astrogliosis.
In summary, the present study has characterized early cell-autonomous mechanisms of astrocyte dysfunction associated with two of the most prevalent SOD1 mutations in ALS patients. The described astrocyte phenotypes have the potential to contribute to mechanisms of MN degeneration in ALS, further underscoring the importance of considering astrocyte dysfunction when developing therapies for ALS.
4. Materials and Methods
4.1. Human induced pluripotent stem cells
Human iPSC lines SOD1(A4V) and SOD1(D90A) were obtained from Target ALS (https://www.targetals.org; Cat No. ND35671 (A4V) and ND35660 (D90A). To generate matching isogenic control iPSC lines Iso(A4V) and Iso(D90A) from the parental lines, CRISPR editing was performed using established methods (Deneault et al., 2021). All iPSC lines were maintained at the Montreal Neurological Institute-Hospital through procedures conducted under Ethical Review Board approval by the McGill University Health Centre Board (DURCAN_IPSC/2019-5374). Undifferentiated state of iPSCs was assessed by testing for expression of the stem cell markers NANOG and OCT4 using rabbit anti-NANOG (1/1,000; Abcam; Cambridge, UK; Cat. No. ab21624) and rabbit anti-OCT4 (1 μg/ml; Abcam; Cat. No. ab19857) or goat anti-OCT3/4 (1/500; Santa Cruz Biotechnology; Dallas, TX, USA, Cat. No. sc-8628) antibodies, and by quality control profiling as described previously (Chen et al., 2021).
4.2. Derivation of neural progenitor cells from human iPSCs
Human iPSCs at low passage number were cultured in mTeSR medium (STEMCELL Technologies; Vancouver, BC, Canada; Cat. No. 85850) in 10-cm culture dishes (Thermo-Fisher Scientific; Waltham, MA; Cat. No. 353003) coated with Matrigel (Thermo-Fisher Scientific; Cat. No. 08-774-552) until they reached 70%-80% confluence. To generate NPCs, iPSCs were dissociated with Gentle Cell Dissociation Reagent (STEMCELL Technologies; Cat. No. 07174), followed by seeding of 2-3×106 cells onto T25 flasks (Thermo-Fisher Scientific; Cat. No. 12-556-009) coated with Matrigel. Cells were then cultured overnight with 5 ml mTeSR supplemented with 10 μM ROCK inhibitor (compound Y-27632 2HCl; Selleck Chemicals; Houston, TX, USA; Cat. No. S1049). At in vitro day 1 (DIV1), mTeSR was replaced with ‘neural induction medium’ containing DMEM/F12 supplemented with GlutaMax (1/1; Thermo-Fisher Scientific; Cat. No. 10565-018), Neurobasal medium (1/1; Thermo-Fisher Scientific; Cat. No. 21103-049), N2 (0.5X; Thermo-Fisher Scientific; Cat. No. 17504-044), B27 (0.5X; Thermo-Fisher Scientific; Cat. No. 17502-048), ascorbic acid (100 μM; Sigma-Aldrich; St. Louis, MO, USA; Cat. No. A5960), L-Glutamax (0.5X; Thermo-Fisher Scientific; Cat. No. 35050-061), antibiotic-antimycotic (1X; Thermo-Fisher Scientific; Cat. No. 15240-062), 3 μM CHIR99021 (STEMCELL Technologies; Cat. No. 72054), 2 μM DMH1 (Sigma-Aldrich; Cat. No. D8946), and 2 μM SB431542 (Tocris Bioscience; Bristol, UK; Cat. No. 1614). The culture medium was changed every other day until DIV6, when induced NPCs were instructed to acquire a caudalized and ventralized progenitor cell identity as follows. NPCs were dissociated with Gentle Cell Dissociation Reagent and split 1:6 with the same medium described above, supplemented with retinoic acid (RA) (0.1 μM; Sigma-Aldrich; Cat. No. R2625) and purmorphamine (0.5 μM; Sigma-Aldrich; Cat. No. SML-0868) in combination with 1 μM CHIR99021, 2 μM DMH1 and 2 μM SB431542 reagents. The culture medium was changed every other day until DIV12, when cells were split again 1:6 and expanded with the same medium containing 3 μM CHIR99021, 2 μM DMH1, 2 μM SB431542, 0.1 μM RA, 0.5 μM purmorphamine, and 500 μM valproic acid (VPA; Sigma-Aldrich; Cat. No. P4543) till DIV18. The ensuing caudalized and ventralized NPCs were validated by real-time polymerase chain reaction (RT-PCR) and immunocytochemistry.
4.3. Differentiation of astrocytes from human iPSC-derived neural progenitor cells
Induced caudalized/ventralized NPCs were differentiated into astrocytes starting at DIV18 using a defined medium. NPCs were seeded at low cell density (15,000 cells/cm2) in two T25 flasks in the presence of 5 ml of NPC expansion medium containing ROCK inhibitor. Next day, medium was replaced with ‘Astrocyte Differentiation Medium 1’ [ScienceCell Astrocyte Growth Medium (ScienCell Research Laboratories; Carlsbad, CA, USA; Cat. No. 1801b) containing astrocyte growth supplement (ScienCell Research Laboratories; Cat. No. 1852), 1% fetal bovine serum (FBS) (ScienCell Research Laboratories; Cat. No. 0010), 50 U/ml penicillin G, 50 mg/ml streptomycin]. Cells were split 1:4 every week and maintained under these culture conditions for 30 days. Half medium was replaced with fresh medium every 3 to 4 days. At DIV50, cultures were switched to ‘Astrocyte Differentiation Medium 2’ (same as Astrocyte Differentiation Medium 1 but lacking FBS). Induced astrocytes were validated by immunocytochemistry, RT-PCR, and by measuring their response to treatment with a cocktail of IL-1α, TNF-α, and C1q (Liddelow et al., 2017).
4.4. Characterization of induced cells by immunocytochemistry
Induced human NPCs and astrocytes were analyzed by immunocytochemistry, which was performed as described previously (Methot et al., 2018). The following primary antibodies were used: rabbit anti HOXA5 (1/67000; kindly provided by Dr. Jeremy Dasen, New York, University School of Medicine), mouse anti-NKX6.1 (1/500; DSHB; Iowa City, IA; Cat. No. F55A10), mouse anti-GFAP (1/1,000; Sigma-Aldrich; Cat. No. G3893); mouse anti-S100B (1/500; Sigma-Aldrich; Cat. No. S2532), mouse anti-γH2AX (1/500; Millipore; Burlington, MA; Cat. No. 05636), rabbit anti-SOD1 (1/500; Enzo Life Sciences; Farmingdale, NY; Cat. No. ADI-SOD-100-F). Secondary antibodies against primary reagents raised in various species were conjugated to Alexa Fluor 555, Alexa Fluor 488 (1/1,000; Invitrogen; Burlington, ON, Canada). Actin polymerization was visualized by staining of F-actin using Alexa-Fluor-488 phalloidin (1/500; Thermo Fisher; Cat. No. A12379). Images were acquired with a Zeiss Axio Observer Z1 Inverted Microscope using 20X magnification (N.A 0.8) and a ZEISS Axiocam 506 mono camera.
4.5. Characterization of induced cells by real-time polymerase chain reaction
RNA extraction and real-time polymerase chain reaction (RT-PCR) were performed as described (Soubannier et al., 2020). Analysis of gene expression was conducted using the following oligonucleotide primers: Taqman probes C1s, Hs00156159_m1; C3, Hs00163811_m1; CCL2, Hs00234140_m1; CXCL10, Hs00171042_m1; S100A10, Hs00237010_m1; PTX3, Hs00173615_m1; SERPING1, Hs00163781_m1. Primer/probe sets were obtained from ThermoFisher Scientific. Data were normalized with BETA-ACTIN and GAPDH (ACTB Hs01060665_g1; GAPDH Hs02786624_g1). Relative quantification (RQ) was estimated according to the ΔCt method (Schmittgen and Livak, 2008).
4.6. Quantification of protein levels
Conditioned media collected from astrocytes treated, or not, with TNF-α (30 ng/ml), IL-1α (3 ng/ml), and C1q (400 ng/ml) for 48 hr were centrifuged at 300 x g for 5 min and supernatant was recovered. Supernatants were sent for proteomics analysis to SomaLogic Inc., Boulder, Colorado (https://somalogic.com/). For studies comparing conditioned media from cultures of Iso(A4V) and SOD1(A4V) astrocytes, protein levels were analyzed using Bio-Plex Pro Human Cytokine Screening Panel (48-Plex) (Cat. No. 12007283), Bio-Plex Pro Human Chemokine Panel (40-Plex) (Cat. No. 171AK99MR2), and Bio-Plex Pro Human Inflammation Panel 1 (37-plex) (Cat. No. 171AL001M) from Bio-Rad (Hercules, CA).
4.7. Quantification of reactive oxygen species
Cells were incubated in presence of either 1 μM or 5 μM MitoSoxTM (Invitrogen; Cat. #M36008) for 30 minutes in medium containing Hoechst (1/2,000 dilution). Following the incubation period, cells were washed for 5 minutes with 37°C-preheated astrocyte growth medium containing astrocyte growth supplement and 1% FBS. Subsequently, the cell culture medium was replaced once again, and the cells were subjected to microscopic observation. For nuclear MitoSox intensities quantification, regions of interest (ROIs) were obtained from the channel corresponding to the Hoechst staining through thresholding. Each ROI corresponding to the nucleus was then used to measure the mean intensity signal in the MitoSox fluorescence channel. Several pictures corresponding to at least 5,000 cells were counted.
Authorship contribution statement
VS performed all cell culture and microscopy experiments, data analysis, and figure preparation. MC, LG, SL, DK, GH performed experiments. SS, TMD, VS, GR conceived overall study plan. SS, TMD supervised the study. SS and VS wrote the manuscript.
Declaration of competing interests
The authors declare no competing interests.
Funding
S.S. and G.R. were supported by funding from ALS Canada/Brain Canada Hudson Translational Team Grant. T.M.D. received funding to support this project through the Canada First Research Excellence Fund, awarded through the Healthy Brains, Healthy Lives initiative at McGill University and an ALS Canada/Brain Canada Discovery grant. S.S. is a Distinguished James McGill Professor of McGill University.
Acknowledgments
We thank Anna Kristyna Franco Flores for experimental assistance, and Valerio Piscopo for discussions and advice.