Research ArticleAmyotrophic Lateral Sclerosis

Genetic validation of a therapeutic target in a mouse model of ALS

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Science Translational Medicine  06 Aug 2014:
Vol. 6, Issue 248, pp. 248ra104
DOI: 10.1126/scitranslmed.3009351

Abstract

Neurons produced from stem cells have emerged as a tool to identify new therapeutic targets for neurological diseases such as amyotrophic lateral sclerosis (ALS). However, it remains unclear to what extent these new mechanistic insights will translate to animal models, an important step in the validation of new targets. Previously, we found that glia from mice carrying the SOD1G93A mutation, a model of ALS, were toxic to stem cell–derived human motor neurons. We use pharmacological and genetic approaches to demonstrate that the prostanoid receptor DP1 mediates this glial toxicity. Furthermore, we validate the importance of this mechanism for neural degeneration in vivo. Genetic ablation of DP1 in SOD1G93A mice extended life span, decreased microglial activation, and reduced motor neuron loss. Our findings suggest that blocking DP1 may be a therapeutic strategy in ALS and demonstrate that discoveries from stem cell models of disease can be corroborated in vivo.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting both lower and upper motor neurons in the brain and spinal cord. To date, riluzole is the only U.S. Food and Drug Administration–approved therapy for ALS (1). Unfortunately, riluzole’s beneficial effects are limited, necessitating the need for further drug discovery efforts. ALS cases are predominantly sporadic; however, concerted effort has allowed the identification of multiple genes involved in both familial and sporadic forms of the disease. Patients have been found to harbor mutations in SOD1 (superoxide dismutase 1), TDP43 (TAR DNA binding protein 43), FUS (fused in sarcoma), UBQLN, OPTN, VCP, and C9ORF72 (210). Although efforts to develop mouse models for these many mutations are under way, currently only the SOD1 mouse model is widely used (1115).

Mutations in the SOD1 gene predominantly cause ALS in a dominant manner. It is thought that a gain of toxic function leads to motor neuron degeneration in these cases. However, a complete understanding of how mutant SOD1 causes disease remains elusive. Mutant SOD1 mouse models have revealed several molecular pathways involved in the process of motor neuron degeneration. Some of these pathways have been proposed to act specifically in motor neurons, including mitochondrial dysfunction, activation of the endoplasmic reticulum stress response, and protein aggregation. In contrast, other disease mechanisms have been suggested to change the function of neighboring glia cells, which in turn lead to motor neuron dysfunction and degeneration. Specifically, there has been increasing interest in understanding how pathological changes in astrocytes, microglia, and oligodendroglial progenitors contribute to ALS [reviewed by Boillée et al. (16)].

The ability to generate motor neurons from embryonic stem cells (ESCs) offers an attractive system for dissecting nonautonomous effects of various cell types on neural degeneration. This ready supply of motor neurons allows them to be placed into coculture with various cell types implicated in disease processes, allowing their interactions to be studied. In particular, this approach has been used to demonstrate that astrocytes and microglia produce substances that are toxic to motor neurons (1723). Conditional deletion of the disease-causing SOD1 transgenes from these glial cell types in vivo substantially increased life span in the ALS mouse model, providing important further evidence that these cells contribute to neuronal degeneration (2426).

Although a combination of in vitro cell culture and in vivo genetic studies has firmly established a role for glial cells in motor neuron degeneration, agreement on the mechanisms through which they act has been slow to develop. Validation of candidate mechanisms in the SOD1G93A mouse model would aid in the generation of consensus and accelerate drug discovery. More broadly, it remains unclear how well mechanistic insights identified using the ever-growing repertoire of stem cell models for neurological conditions will translate into animal models (1821, 2739).

Here, we demonstrate that elimination of the DP1 receptor reduces the toxicity of SOD1G93A microglia to motor neurons and extends life span in the SOD1G93A animal model of ALS. Thus, our results firmly establish inhibition of the DP1 receptor as a therapeutic target for ALS and more generally support the notion that mechanistic insights derived from stem cell models can be validated in vivo.

RESULTS

Previously, we have shown that treatment of control glial populations with prostaglandin D2 (PGD2) resulted in a toxicity to motor neurons that was similar to that observed when motor neurons were cocultured with primary glia from the SOD1G93A mouse model (18). However, we had not resolved whether either of the PGD2 receptors, DP1 or DP2, was responsible for mediating the resulting glial toxicity. Furthermore, the primary glial cell preparations used for our previous study contained a variety of cell types, including microglia and astrocytes, and it was therefore unclear which of these cell types were responding to initiate motor neuron degeneration (fig. S1A). Because of the availability of both a selective small-molecule agonist and antagonist of DP1, we opted to first investigate whether DP1 plays an essential role in mediating glial toxicity to motor neurons.

The compound MK0542 is known to modestly inhibit both DP1 and DP2, whereas BW A868C is thought to be a potent and selective inhibitor of DP1 (Fig. 1, A and B) (40). Therefore, to begin resolving whether the DP1 receptor plays a role in glia toxicity, we tested whether these compounds could protect motor neurons from SOD1G93A glial toxicity. To test this idea, we used a model of glial toxicity to stem cell–derived human motor neurons similar to that we previously reported (18). In brief, HUES3 Hb9::green fluorescent protein (GFP) ESCs were differentiated into motor neurons using a 24-day protocol (41, 42). These motor neurons were then purified on the basis of GFP expression by fluorescent-activated cell sorting (FACS) and cocultured with either nontransgenic or SOD1G93A primary glial monolayers isolated from neonatal mice. After 10 days of coculture, the number of GFP+ motor neurons was quantified (Fig. 1C). We noted that the glial monolayers we used for these studies consisted of GFAP+ (glial fibrillary acidic protein–positive) astrocytes and CD11b+ microglia (fig. S1A).

Fig. 1. DP1 plays a role in glial toxicity to motor neurons.

(A) Chemical structures of MK0542 (a dual DP antagonist), BW A868C (a DP1 antagonist), and BW 245C (a DP1 agonist). (B) Prostaglandin pathway. (C) Experimental design of the coculture system. (D) Percentage of Hb9::GFP+ human motor neurons 10 days after plating on SOD1G93A or nontransgenic (No-Tg) mouse glia treated with vehicle [dimethyl sulfoxide (DMSO)], MK0524, or BW A868C (mean ± SEM; n = 3). (E) Percentage of Hb9::GFP+ human motor neurons after 10 days of coculture with nontransgenic glia treated with vehicle (DMSO) or BW 245C (mean ± SEM; n = 5). (F) Human neuronal cells after 10 days of coculture with nontransgenic mouse glia. Arrows indicate human motor neurons, MAP2+/Hb9::GFP+ cells; asterisks indicate MAP2+/Hb9::GFP cells, nonmotor neurons. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (G) Percentage of MAP2+ neurons after 10 days of coculture with nontransgenic glia treated with DMSO or BW 245C (mean ± SEM; n = 2). Statistical significance was analyzed using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Similar to previous studies, 10-day coculture of purified motor neurons with mutant SOD1G93A glia led to a 55% decrease in motor neuron survival relative to coculture with control glia (Fig. 1D). Treatment with the dual DP antagonist MK0542 provided a modest protection from this toxic effect (Fig. 1D). Strikingly, chronic treatment with the selective DP1 antagonist BW A868C completely protected motor neurons from SOD1G93A glia, returning rates of survival to control levels (Fig. 1D). Dose-response experiments demonstrated that BW A868C abrogated glial toxicity at and above its Ki (inhibition constant) for DP1 (1 nM) (fig. S1B). Lower concentrations were ineffective, suggesting a specific effect (fig. S1B).

Reciprocally, we reasoned that if the DP1 receptor was indeed modulating glial toxicity, then a selective chemical activator of DP1 such as BW 245C (43, 44) might render control glia selectively toxic to motor neurons (Fig. 1, E to G). We treated cocultures of purified motor neurons and control nontransgenic glia with either vehicle (DMSO) or DP1 agonist (BW 245C) for 10 days. After DP1 agonist treatment, we observed a 49% reduction in motor neuron number relative to vehicle, which was similar to the toxic effect of SOD1G93A glia (Fig. 1E).

We next asked if the toxic effect induced by the DP1 agonist was specific to motor neurons. We performed motor neuron differentiation with HUES3 Hb9::GFP and, rather than purifying GFP+ motor neurons, cultured the resulting mixture of motor neurons and other neural types with control glia, in the presence of either vehicle or DP1 agonist. We then quantified the total number of neurons using immunostaining with antibodies specific to MAP2 and counted motor neuron number by GFP fluorescence. We again found a decrease in the number of MAP2+/Hb9::GFP+ motor neurons after treatment with the DP1 agonist (Fig. 1, F and G). In contrast, the number of MAP2+/Hb9::GFP neurons, which were predominantly nonmotor neurons, remained unchanged (Fig. 1, F and G). Thus, treatment with the DP1 agonist (BW 245C) induced nontransgenic control glia to behave similarly to SOD1G93A glia and selectively induced death of motor neurons, but not death of other neurons.

To explore the downstream effect of these compounds on glia, we analyzed adenosine 3′,5′-monophosphate (cAMP) concentrations. It has been reported that when BW A868C binds the DP1 receptor in rabbit epithelial cells and bovine fibroblasts, cAMP concentrations were decreased (45, 46). Reciprocally, BW 245C raises cAMP concentrations in various cell types (4649). We therefore wished to determine whether these compounds had similar effects on our glial preparations. As predicted by previous results, when SOD1G93A glia were treated with a DP1 antagonist (BW A868C), cAMP concentrations were decreased (fig. S1C). Additionally, when nontransgenic control glia were treated with a DP1 agonist (BW 245C), cAMP concentrations were increased (fig. S1D). Together, these results were consistent with the notion that the negative effects of SOD1G93A glia were at least in part mediated through the DP1 receptor.

DP1 acts in glia to modulate their toxicity to motor neurons

Given that the DP1 receptor is widely expressed (44, 5053), we went on to address whether DP1 acts in the motor neurons or in glia to modulate their pathological interaction. To discriminate between an interaction of the compounds with either glia or motor neurons, we developed a variation of our coculture system. Nontransgenic or SOD1G93A glia were pretreated with vehicle (DMSO) or compound in the absence of motor neurons for 48 hours. After 48 hours, glial cells were washed twice. Purified motor neurons were then added to the coculture and quantified 10 days later (Fig. 2A). In addition to discriminating between whether a chemical compound was acting on the glia or motor neurons, this assay also allowed us to determine whether ongoing DP1 inhibition was required for neuronal protection or, instead, if DP1 inhibition induced a long-lasting change in glial cells. When SOD1G93A glial cells were pretreated with vehicle, these cells induced motor neuron loss in a manner similar to our standard assay (Fig. 2, B and D). However, transient exposure of SOD1G93A glia to the DP1 antagonist for 48 hours before coculture with motor neurons significantly reduced their toxicity (P = 0.004) (Fig. 2, B and D). Reciprocally, a transient treatment of control glia with the DP1 agonist (BW 245C) induced a loss of motor neurons during subsequent coculture, even though the agonist had been washed away before addition of the motor neurons (Fig. 2, C and E). In contrast, when motor neurons were purified, plated in isolation, and then treated with the DP1 agonist (BW 245C), motor neuron survival was not affected (fig. S2E).

Fig. 2. DP1 acts in glia to modulate their toxicity to motor neurons.

(A) Experimental design of the pretreatment coculture system. (B) Percentage of Hb9::GFP+ cells after 10 days of coculture with pretreated nontransgenic or SOD1G93A mouse glia with either vehicle (DMSO) or DP1 antagonist (BW A868C) (mean ± SEM; n = 3). (C) Percentage of Hb9::GFP+ cells after 10 days of coculture with nontransgenic mouse glia pretreated with vehicle or BW 245C (mean ± SEM; n = 3). (D) Images of Hb9::GFP+ cells after 10 days of coculture with pretreated nontransgenic or SOD1G93A glia with either vehicle (DMSO) or DP1 antagonist (BW A868C). (E) Images of Hb9::GFP+ cells after 10 days of coculture with pretreated nontransgenic glia with either vehicle (DMSO) or DP1 agonist (BW 245C). Scale bars, 100 μm. Statistical significance was analyzed using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

These experiments allowed us to draw two conclusions concerning the likely role of the DP1 receptor in mediating glial toxicity to motor neurons. First, the DP1 antagonist and agonist influenced glial toxicity rather than acting directly on motor neurons. Second, transient modulation of the DP1 receptor in glia induced a long-lasting change in their phenotype.

DP1 mutant glia are less toxic to motor neurons

It is well known that chemical compounds can have off-target effects. To rule out such off-target effects in this context, we used mice harboring a targeted mutation in the Ptgdr gene, which encodes the DP1 receptor (Fig. 3A and fig. S2, A and B) (51). We crossed this targeted Ptgdr (DP1) mutation into the SOD1G93A transgenic background. We then prepared glial cultures from DP1+/+, DP1+/−, and DP1−/− mice, both with and without the SOD1G93A transgene (Fig. 3A).

Fig. 3. DP1 mutant glia are less toxic to motor neurons.

(A) Schematic overview of matings between different transgenic mouse strains. (B to F) Relative expression of genes in nontransgenic and SOD1G93A glia with differential DP1 expression. (G) Percentage of Hb9::GFP+ cells after 10 days of coculture with nontransgenic or SOD1G93A glia with differential DP1 expression (mean ± SEM; n = 3). (H) Percentage of Hb9::GFP+ cells after 10 days of coculture with nontransgenic glia with differential DP1 expression treated with either vehicle (DMSO) or DP1 agonist (BW 245C) (mean ± SEM; n = 3). Statistical significance was analyzed using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

To study how DP1 inhibition changed the toxic phenotype of glia, we quantified transcript levels of genes involved in an inflammatory phenotype. As expected, the targeted mutation led to a dose-dependent reduction in DP1 transcript levels on both the nontransgenic and SOD1G93A backgrounds (Fig. 3B). Additionally, genetic elimination of DP1 reduced transcript levels of Ptgs2 [cyclooxygenase-2 (Cox-2)] (P = 0.002), Ptgds [lipocalin-type PGD synthases (L-PGDs)] (P = 0.003), and Gpr44 (DP2) (P = 0.007), whereas Ptgs1 (Cox-1) did not show a significant change (P = 0.12) (Fig. 3, C to F). Thus, the protective effect of genetic elimination of DP1 was associated with reduced transcript levels of genes involved in a proinflammatory phenotype.

We next cocultured mouse glia of varying SOD1G93A and DP1 mutant compound genotypes with purified human motor neurons and monitored their survival. This experiment revealed a dose-dependent reduction in toxicity to motor neurons upon genetic removal of Ptgdr (DP1) in SOD1G93A glia (Fig. 3G and fig. S2C). Removing one allele of Ptgdr (DP1) reduced the toxicity of SOD1G93A glia, improving motor neuron survival from 52 to 83% (P = 0.02); removing both alleles further reduced their toxic effect on motor neurons, improving motor neuron survival from 52 to 97% (P = 0.0007) (Fig. 3G and fig. S2C). Eliminating one or both alleles of Ptgdr (DP1) from control nontransgenic glia had no effect on motor neuron survival (Fig. 3G and fig. S2C).

The availability of glia from Ptgdr (DP1) mutant animals also afforded us the opportunity to determine whether the effects of the DP1 antagonist (BW A868C) and the DP1 agonist (BW 245C) were selectively mediated through the DP1 receptor. To investigate whether the DP1 receptor was required for the toxicity-inducing activity of the DP1 agonist (BW 245C), we examined its effect on nontransgenic glia with varying Ptgdr (DP1) genotypes cocultured with purified motor neurons. As we described above (Fig. 1), addition of the DP1 agonist to cocultures containing glia derived from nontransgenic animals with a DP1+/+ genotype induced toxicity to motor neurons (Fig. 3H). In contrast, cocultures containing DP1+/− glia showed significantly decreased toxicity to motor neurons after agonist treatment (P = 0.006) (Fig. 3H). When DP1−/− glial cocultures were treated with the DP1 agonist, they displayed no appreciable toxicity to motor neurons (Fig. 3H). Therefore, the toxicity-inducing effect of the DP1 agonist was mediated through the DP1 receptor.

We next determined whether the protective effects of the DP1 antagonist were also selectively mediated through the DP1 receptor. When compound transgenic SOD1G93A/DP1+/− glia were cocultured in the presence of the DP1 antagonist BW A868C, the modest motor neuron toxicity in this setting was rescued (fig. S2D). However, treatment with the antagonist did not provide protection above and beyond completely eliminating the DP1 receptor (SOD1G93A/DP1−/−), therefore ruling out a nonspecific neurotrophic or supportive effect of the DP1 antagonist (fig. S2D). Overall, our genetic studies demonstrated that DP1 is an important modulator of both prostanoid signaling pathway transcription and in vitro glial toxicity.

A DP1 mutation extends life span in SOD1G93A mice

It is still unclear to what extent findings from stem cell models will be predictive of outcomes in vivo. Therefore, we tested whether genetic elimination of DP1 could extend life span in the SOD1G93A mouse model of ALS (1719). To this end, we created compound SOD1G93A transgenic/Ptgdr (DP1) mutant mice. We then monitored disease onset, progression, and overall survival in this cohort of animals. In SOD1G93A/DP1+/+ animals, we observed that disease onset as measured by a sustained decrease in body weight occurred at 106 ± 4 days (fig. S3E). Genetic elimination of one or both alleles of Ptgdr (DP1) did not significantly alter the time of disease onset (SOD1G93A/DP1+/−, 108 ± 2 days; SOD1G93A/DP1−/−, 111 ± 3 days) (fig. S3E). However, both SOD1G93A/DP1+/− and SOD1G93A/DP1−/− animals lived significantly longer than mice with an SOD1G93A/DP1+/+ genotype [SOD1G93A/DP1+/+, 134 ± 2 days; SOD1G93A/DP1+/−, 145 ± 2 days, P = 0.0002; SOD1G93A/DP1−/−, 143 ± 2 days, P = 0.0176 (log-rank test)] (Fig. 4, A and B). This change in average life span translated into a 6.7% increase in total life span of SOD1G93A/DP1−/− animals and an 8.2% increase in the life span of SOD1G93A/DP1+/− animals.

Fig. 4. DP1 mutations extend life span and progression in SOD1G93A mice.

(A) Cumulative probability of survival, shown in a Kaplan-Meier plot (control DP1+/+ SOD1G93A, red, n = 25; DP1+/− SOD1G93A, purple, n = 56; DP1−/− SOD1G93A, blue, n = 27). (B) Mean survival of different genotypes (mean ± SEM). (C) Weight at weaning in grams of DP1−/− animals compared to DP1+/− and DP1+/+ animals (both nontransgenic and SOD1G93A) (mean ± SEM, n = 10). (D) Average number of choline acetyltransferase (ChAT)–positive cells per spinal cord section (mean ± SEM, n = 3). (E) Relative expression of Hb9 in nontransgenic or SOD1G93A mouse spinal cords (mean ± SEM; n = 3 for day 100; n = 4 for end stage). (F to H) Representative images from ChAT-positive cells in mouse lumbar spinal cord from DP1+/+ SOD1G93A, DP1+/− SOD1G93A, or DP1−/− SOD1G93A mice at day 100. Scale bars, 100 μm. Statistical significance was analyzed using one-way analysis of variance (ANOVA) and Mantel-Cox log-rank test. *P < 0.05, **P < 0.01, ***P < 0.001.

Consistent with the idea that glial toxicity is important in the progressive phase of the disease, there was also a statistically significant lengthening of the progressive phase of the disease in SOD1G93A/DP1+/− animals [26 ± 4 days in SOD1G93A/DP1+/+ mice, 38 ± 2 days in SOD1G93A/DP1+/− mice, P = 0.0186 (log-rank test)] (fig. S3F). We found that the increased life span of SOD1G93A/Ptgdr (DP1) mutant mice was not the result of a trivial decrease in SOD1G93A copy number in these animals (fig. S3, B to D).

We noted that unlike in our in vitro stem cell model, where complete elimination of the DP1 receptor in the SOD1G93A genetic background provided additional motor neuron protection beyond that found in SOD1G93A/DP1+/− glia, life span extension in SOD1G93A/DP1−/− and SOD1G93A/DP1+/− littermates was similar. A lifelong decrease in body weight of control nontransgenic DP1−/− animals relative to their DP1+/− and DP1+/+ littermates might be explanatory (Fig. 4C and fig. S3A). In both animal models and ALS patients, it has been shown that future survival is affected by body weight at disease onset. The significantly decreased body weight of DP1−/− mutant mice therefore may have masked additional protection by the complete genetic elimination of DP1. Yet, survival was significantly extended by the genetic reduction of DP1 in both SOD1G93A/DP1+/− and SOD1G93A/DP1−/− animals compared to their SOD1G93A/DP1+/+ counterparts, thus providing in vivo validation that DP1 suppression is a relevant therapeutic strategy in ALS.

DP1 mutation protects motor neurons and decreases microglia activation in vivo

As our culture model had indicated that elimination of DP1 reduced glial toxicity, we reasoned that extension of life span in SOD1G93A/DP1+/− and SOD1G93A/DP1−/− animals might also be associated with an increased survival of motor neurons and changes in astrocytes or microglia. To study the influence of genetic modulation of DP1, spinal cords were harvested at day 100 and at the end stage of disease from SOD1G93A animals of varying DP1 genotypes. We then analyzed the number of motor neurons per spinal cord section with anti-ChAT antibody staining. We found that SOD1G93A/DP1+/− and SOD1G93A/DP1−/− animals exhibited increased motor neuron survival relative to animals with the SOD1G93A/DP1+/+ genotype (Fig. 4, D and F to H, and fig. S3, G to I). Additionally, when we examined the transcript abundance from motor neuron marker gene Hb9 in these spinal cords, it was reduced in SOD1G93A control (DP1+/+) animals but not in compound DP1 mutant animals (DP1−/−, DP1+/−) (P = 0.005) (Fig. 4E).

We then examined whether this increased survival of motor neurons correlated with changes in microglia and astrocyte behavior. We analyzed microglial activation through immunostaining with anti-CD11b, anti-CD68, and anti-Iba1 antibodies and astrocyte activation with anti-GFAP antibody (Fig. 5 and figs. S3, J to L, and S4). We did not observe a qualitative change in GFAP expression in SOD1G93A transgenic animals that correlated with their DP1 genotype (fig. S3, J to L). However, we did find that SOD1G93A/DP1+/− and SOD1G93A/DP1−/− animals exhibited fewer CD68+ and Iba1+ microglia in the spinal cord than their counterparts with a SOD1G93A/DP1+/+ genotype (Fig. 5, A to C).

Fig. 5. DP1 mutations decrease microglia activation.

(A) Percentage of CD11b+ cells per spinal cord section relative to expression in DP1+/+ SOD1G93A mouse spinal cord (mean ± SEM, n = 3). CD11b+ cells in lumbar spinal cord at day 100 of DP1+/+ SOD1G93A, DP1+/− SOD1G93A, or DP1−/− SOD1G93A mice. (B) Percentage of Iba1+ cells per spinal cord section relative to expression in DP1+/+ SOD1G93A mouse spinal cord (mean ± SEM, n = 3). Iba1+ cells in lumbar spinal cord at day 100 from DP1+/+ SOD1G93A, DP1+/− SOD1G93A, or DP1−/− SOD1G93A mice. (C) Percentage of CD68+ cells per spinal cord section relative to expression in DP1+/+ SOD1G93A mouse spinal cord (mean ± SEM, n = 3). CD68+ cells in lumbar spinal cord from DP1+/+ SOD1G93A, DP1+/− SOD1G93A, or DP1−/− SOD1G93A mice at day 100. (D to F) Relative expression of genes in nontransgenic or SOD1G93A mouse spinal cord (mean ± SEM; n = 3 for day 100; n = 4 for end stage). Scale bars, 100 μm. Statistical significance was analyzed using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

To determine how these observations correlated with gene expression, we analyzed transcript levels of these genes in the spinal cord at day 100 and at end stage. At day 100, we found that transcript levels of CD11b were decreased in compound SOD1G93A/DP1 mutant animals (DP1−/−, DP1+/−) compared to SOD1G93A control (DP1+/+) animals (P = 4.67*10–5) (Fig. 5E). Although it was not evident by immunostaining, we also found a decrease in GFAP transcript abundance (Fig. 5D). Additionally, end-stage transcript levels of CD68, another marker of activated microglia, were significantly reduced in SOD1G93A/DP1 mutant animals (DP1−/−, DP1+/−) compared to SOD1G93A control (DP1+/+) animals (P = 0.007) (Fig. 5F). Furthermore, when we stained sections of spinal cords at end stage with CD11b, CD68, and Iba1 antibodies, in each case, we found fewer immunopositive microglia in SOD1G93A DP1+/− than in SOD1G93A DP1+/+ animals (fig. S4, G to I).

To explore if the decrease in microglial activation was specifically related to DP1 inhibition in SOD1G93A animals or if nontransgenic DP1 mutant animals also expressed reduced microglia activation, we analyzed CD11b transcript levels in nontransgenic DP1+/+, DP1+/−, and DP1−/− animals and found no significant difference between the three DP1 genotypes (fig. S4A). Additionally, we wondered if the changes we found in gene expression levels of proinflammatory markers in glia in vitro (Fig. 3) were comparable to changes in expression in spinal cords of the animals with various DP1 genotypes. Transcript levels of Ptgdr (DP1), Ptgs1 (Cox-1), Ptgs2 (Cox-2), and Ptgds (L-PGDs) were significantly reduced in SOD1G93A DP1 mutant animals (DP1−/−, DP1+/−) when compared to SOD1G93A control (DP1+/+) animals, whereas Gpr44 (DP2) did not show a significant change (fig. S4, B to F).

These results suggest that even when variation in total life span was taken into account by performing our analyses at disease end stage, DP1 mutant animals still showed a reduced inflammatory expression profile. Furthermore, DP1 reduction resulted in decreased expression of the inflammatory markers Ptgdr (DP1), Ptgs2 (Cox-2), and Ptgds (L-PGDs). This observation held true both in vitro, in SOD1G93A glia with genetic reduction of DP1, and in vivo, in the spinal cord in DP1 mutant SOD1G93A animals (Fig. 3, B, E, and F, and fig. S4, B, E, and F). Thus, DP1 reduction resulted in a similar decrease in expression of inflammatory markers both in vitro and in vivo.

DP1 modulates microglial toxicity to motor neurons

Although we saw strong evidence for a reduced microgliosis in SOD1G93A/DP1 mutant animals, we also saw modest evidence for a decline in astrogliosis as shown by a decrease in GFAP transcript levels. We therefore returned to our in vitro system to test if DP1 was acting in microglial, astroglial, or both cell types to modulate their toxicity to motor neurons (Fig. 6).

Fig. 6. Microglia toxicity in coculture assays.

(A) Experimental setup for purified mouse astrocytes and human motor neuron cocultures. Astrocytes were purified and cultured for 72 hours, motor neurons were added, and cocultures were treated with DMSO or compounds. Survival was analyzed after 10 days. (B) Percentage of Hb9::GFP+/MAP2+ motor neurons after coculture with purified astrocytes, treated with DMSO or BW A868C. (C) Percentage of Hb9::GFP/MAP2+ motor neurons after coculture with purified astrocytes, treated with DMSO or BW 245C. N.S., not significant. (D) Experimental setup of microglia spike-in assay. Mouse microglia were purified, pretreated for 48 hours with DMSO or compounds, and then washed and added onto 1-day-old cultures of human motor neurons. Survival was analyzed after 10 days of coculture. (E) Percentage of Hb9::GFP+ cells after spike-in of microglia pretreated with DMSO or BW A868C. (F) Percentage of Hb9::GFP+ cells after spike-in of microglia pretreated with DMSO or BW 245C. (G) Experimental setup of microglia spike-in assay with various DP1 genotypes. (H) Percentage of Hb9::GFP+ cells after spike-in of microglia derived from various DP1 genotypes. (I) Relative expression of DP1 (Ptgdr) in purified motor neurons, astrocytes, and microglia. Mean ± SEM; n = 3. Statistical significance was analyzed using two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

To investigate if astrocytes were the main cell type exerting the protective effect of DP1 modulation, we created a coculture assay consisting of purified mouse astrocytes and human motor neurons. To obtain purified astrocytes, we performed crosses of the SOD1G93A mice with GFAP::GFP animals, derived glial cultures, and purified GFAP::GFP+ astrocytes by FACS. Astrocytes were plated for 72 hours and then cocultured with motor neurons for an additional 10 days (Fig. 6A). Congruent with previous work, SOD1G93A mutant astrocytes were modestly toxic to motor neurons (Fig. 6B) (17, 19, 20). Notably, this toxicity was not rescued when these cocultures were treated with the DP1 antagonist (BW A868C), suggesting that DP1 modulation in astrocytes was not responsible for the protective effects we observed (Fig. 6B). To analyze if the toxic gain of function induced by the DP1 agonist was mediated through astrocytes, purified GFAP::GFP+, SOD1G93A, control astrocytes were cocultured with motor neurons in the presence of vehicle (DMSO) or agonist (BW 245C). No motor neuron toxicity was observed when purified astrocytes were treated with the DP1 agonist (Fig. 6C).

To address whether microglia were contributing to the motor neuron–toxic environment of our SOD1G93A glial cultures, we purified CD11b+ microglia by FACS from nontransgenic and SOD1G93A glial cultures and then spiked each genotype onto a monolayer of purified motor neurons (Fig. 6D). Relative to nontransgenic microglia, introduction of purified SOD1G93A microglia resulted in significantly decreased motor neuron survival (P = 0.002) (Fig. 6E). Supporting the idea that the DP1 receptor is an important mediator of this toxicity, a transient pretreatment of purified SOD1G93A microglia with the DP1 antagonist (BW A868C) before their addition to cocultures eliminated their toxic activity (Fig. 6E). Reciprocally, when purified nontransgenic microglia were pretreated with the DP1 agonist (BW 245C), a significant decline in motor neuron survival was observed (Fig. 6F). Thus, pharmacological modulation of DP1 in purified microglia was sufficient to modify their toxicity to motor neurons.

We then went on to investigate whether the effects of chemical DP1 modulation on microglia could be mirrored by genetic modulation of DP1. We performed a coculture of motor neurons with purified microglia derived from the cohort of SOD1G93A animals with varying DP1 genotypes (Fig. 6G). As we have shown before, when DP1+/+ SOD1G93A microglia were spiked onto a motor neuron monolayer, a decrease in motor neuron survival was observed compared to spiking with control DP1+/+ microglia (Fig. 6H and fig. S5A). When DP1 mutant SOD1G93A microglia (DP1−/−, DP1+/−) were added to the motor neurons, toxicity to motor neurons was eliminated (Fig. 6H and fig. S5A). Overall, these experiments conclusively show that the effects of DP1 modulation on motor neuron survival can be mediated through microglia.

We next compared the transcript levels of DP1 (Ptgdr) in the cell types used in our assays. Both astrocytes and microglia expressed increased DP1 relative to motor neurons, with SOD1G93A microglia showing further increased DP1 expression (Fig. 6I). To explore how reduced DP1 activity in these SOD1G93A microglia resulted in a nontoxic motor neuron environment, we analyzed the expression of several microglia polarization markers. First, we examined changes in expression between nontransgenic and SOD1G93A microglia (fig. S5, B and C). Then, we analyzed the same markers in vehicle (DMSO)– and DP1 antagonist (BW A868C)–treated SOD1G93A microglia (fig. S5, D and E). When purified microglia were treated for 48 hours with the DP1 antagonist, microglia polarization was induced toward a “protective” M2 state, accompanied by a modest attenuation of the more inflammatory M1 state. We found a significant increase in transcript levels of M2 genes Ym1 (P = 0.009), IL4 (P = 0.014), and IL10 (P = 0.019) and a decrease in the M1 gene CD68 after antagonist treatment (P = 0.04) (fig. S5, D and E). Additional markers—CD206, Arg1, TNF-α, IL1β, and iNOS—were not significantly affected by antagonist treatment (fig. S5, D and E).

DP1 activation in human microglia induces motor neuron toxicity

We next asked if chemical modulation of the DP1 receptor affected how human glial preparations responded to human motor neurons. To this end, we obtained two distinct cultures derived from human fetal brain tissue. The first was a mixed population of fetal cells containing cells with astrocytic properties that had previously been shown to be capable of SOD1G37R-mediated toxicity to human motor neurons (20). The second was a population of purified fetal microglia.

Consistent with previous descriptions of these cells (20), fetal cultures containing astrocytic cells uniformly expressed A2B5 and CD44, with a more modest population (3 to 15%) expressing S100B and GFAP (Fig. 7C and fig. S6A). Fetal microglia were predominantly Iba1+ (72%) with a more modest subset of cells expressing CD11b (15%) and CD68 (6%) (Fig. 7F). We further characterized these human fetal astrocytes through RNA sequencing, which suggested that relative to fibroblasts, they expressed increased levels of several astrocytic markers including GFAP (fig. S6B).

Fig. 7. DP1 activation in human microglia induces motor neuron toxicity.

(A) Experimental setup of human fetal astrocytes and human motor neuron cocultures. Cocultures were treated with DMSO or BW 245C. (B) Percentage of Hb9::GFP+ cells after 10 days of coculture with human fetal astrocytes, treated with DMSO or BW 245C (mean ± SEM; n = 4). (C) Characterization of human fetal astrocytes, GFAP+, and A2B5+ cells. Scale bars, 100 μm. (D) Experimental setup of human fetal microglia and human motor neuron cocultures. Human fetal microglia were pretreated for 48 hours with DMSO or BW 245C. Microglia were washed and added in equal numbers onto motor neurons. (E) Percentage of Hb9::GFP+ cells after 10 days of coculture with human fetal microglia, pretreated with DMSO or BW 245C (mean ± SEM; n = 4). (F) Characterization of human fetal microglia, Iba1+, and CD68+ cells. Scale bars, 50 μm. (G) Relative expression of DP1 in human motor neurons (Mn), astrocytes, and microglia (mean ± SEM; n = 3). WT, wild type. (H) Relative expression of DP1 in SOD1G93A mouse astrocytes and microglia treated with DMSO or BW A868C (mean ± SEM; n = 3). Statistical significance was analyzed using two-tailed Student’s t test. *P < 0.05, ***P < 0.001.

After an initial characterization of these putative glial cells, we asked if they exerted an effect on human motor neurons when treated with the DP1 agonist BW 245C. We cocultured the human fetal brain preparation containing astrocyte-like cells with purified motor neurons in the presence of vehicle or the DP1 agonist (Fig. 7A). After 10 days, cocultures were analyzed, and no motor neuron toxicity was observed (Fig. 7, A and B). In contrast, human microglia pretreated with DP1 agonist became toxic to motor neurons (Fig. 7, D and E).

We finally asked whether expression of SOD1G93A in these human glia cells would change the expression of DP1. Relative to transduction with wild-type SOD1, transduction with SOD1G93A resulted in a significant increase in DP1 expression (Fig. 7G). Additionally, when cells were treated with the DP1 antagonist, DP1 transcript abundance was decreased (Fig. 7H). We also found that forced expression of SOD1G93A in human astrocyte–like cells led to an increase in GFAP, but this was not reduced by treatment with the DP1 antagonist (fig. S6, C and D). When human microglia were treated with the DP1 antagonist, we did see some evidence for reversal of microglia polarization changes induced by SOD1G93A expression, including increased expression of CD206 and reduced abundance of IL1β (fig. S6, E to H).

DISCUSSION

There are now many examples of the successful recapitulation of known phenotypes from patients in stem cell models of disease (1720, 36, 37, 39). However, there have been few attempts to determine whether mechanistic insights gleaned by stem cell disease modeling can be validated in vivo. As a result, the predictive power of stem cell systems remains in question. Here, we report that the DP1 receptor is a critical mediator of glial toxicity to motor neurons in a stem cell model of ALS. Elimination of even a single allele of the gene encoding this receptor significantly extended the life span of the most widely studied ALS mouse model. We found that a decrease in spinal cord microglial activation was associated with the longer life of these animals, further supporting the predictive nature of our in vitro assay.

Positron emission tomography has indicated widespread microglial activation in the brains of living ALS patients (54). Improper regulation of microglial activation can lead to neuronal dysfunction and death (55). Mutant SOD1 transgenic mice display similar microgliosis and inflammation accompanied by elevated levels of proinflammatory cytokines (5661). Suggesting that these changes are of direct relevance to neuronal degeneration, when SOD1 mutant microglia were cocultured with primary rat or human motor neurons, reduced motor neuron survival was observed [Xiao et al. (62) and this study]. Supporting the notion that restoring normal microglial functionality can have beneficial effects in ALS, introduction of normal microglia via bone marrow transplantation into SOD1G93A animals slowed disease progression (63). In total, previous studies of microglia in the ALS mouse model seem to suggest that identifying factors capable of either normalizing microglial function or slowing their toxic activities could be of therapeutic benefit. Here, we have provided evidence that manipulation of the DP1 receptor offers such an opportunity and that inhibition of the DP1 receptor is a rational strategy for slowing the negative impact of microgliosis in ALS.

Consistent with the notion that DP1 may play a broader role in nervous system degeneration, this receptor has also been shown to influence disease progression in the Twitcher mouse. When DP1 was genetically eliminated from this mouse model of demyelination, a decrease in spasticity was observed that was associated with reduced demyelination and gliosis (64). Furthermore, antagonizing DP1 proved to be neuroprotective in an in vitro model of Alzheimer’s disease, whereas DP1 activation through BW 245C induced neuronal damage in this model (65).

It is notable that although survival of SOD1G93A/DP−/− mice surpassed that of DP+/+ animals, it was not extended beyond that seen in DP+/− animals. This might in part be explained by the lifelong decrease in body weight of control nontransgenic DP1−/− animals relative to their DP1+/− and DP1+/+ littermates (Fig. 4, A and C). It is well known that body weight at disease onset is an important prognosticator of future survival in both this animal model and ALS patients (66). Thus, it may be that the significantly decreased body weight of DP1−/− mutant mice masked additional protection from glial toxicity afforded by complete genetic elimination of DP1. Nevertheless, in the SOD1G93A background, genetic reduction of DP1 in both SOD1G93A/DP1+/− and SOD1G93A/DP1−/− animals significantly extended survival relative to their SOD1G93A/DP1+/+ counterparts, providing in vivo validation that DP1 suppression may be a relevant strategy in the context of ALS.

Evidence supporting the notion that a therapeutic benefit might be drawn from DP1 inhibition stretches beyond observations in cell culture and in mice. As we observed in mouse microglia, DP1 was identified as one of several mediators of prostanoid signaling to be expressed at elevated levels in the spinal cord of ALS patients (67).

Finally, it is also noteworthy that DP1 is a G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor and therefore a rational drug target. Although the chemical compounds used in the assays we report here are not currently in therapeutic development, they did allow us to provide evidence that pharmacological modulation of DP1 can lead to long-lasting effects on glial phenotypes. Furthermore, highly potent and selective DP1 antagonists suitable for clinical study have been developed that could be repurposed for ALS (68). Perhaps most encouraging, our results showing that genetic elimination of a single copy of DP1 can extend life span suggests that even modest pharmacological inhibition might slow disease.

MATERIALS AND METHODS

Study design

The objective of this study was to analyze if DP1 inhibition leads to an increase in survival of ALS animals and how this protective effect is mediated both in vivo and in vitro. We performed a survival study for SOD1G93A animals with varying DP1 genotypes, and the endpoint was determined by the mouse’s inability to right itself within 30 s. Only littermate animals were used to decrease variance that was not due to a decreased expression of DP1 between groups. SOD1G93A copy number was analyzed to ensure that the increased life span of SOD1G93A/Ptgdr (DP1) mutant mice was not the result of a decrease in copy number in these animals. For the in vitro assays, n values reported are independent experimental replicates: Motor neurons were derived from different passages of the HUES3 Hb9::GFP line, and mouse glia cells were derived from different animals. Again, only littermates were used in a direct comparison. Furthermore, in the coculture assays, an n of 1 represents at least three different wells per condition that was analyzed. The n values reported in the human fetal glia experiments represent motor neurons derived from a different passage of HUES3 Hb9::GFP and human fetal glia derived from a different passage of the astrocyte or microglia cell line. Chemical treatment studies were performed on cells and cocultures derived from the same source treated with either DMSO (vehicle) or compound, and subsequent analysis was carried out.

ESC culture and differentiation

All cell cultures were maintained at 37°C, 5% CO2. The HUES3 Hb9::GFP line was maintained on Matrigel (Fisher Scientific, catalog no. 08-774-552; BD Biosciences, catalog no. 354277) in mTeSR medium (STEMCELL Technologies). Medium was changed daily, and cells were passaged using dispase (Invitrogen). ESCs were dissociated with accutase into single cells. Cells were resuspended into mTeSR supplemented with 10 μM Rho-associated kinase inhibitor Y27632 (Sigma) and cultured in ultralow attachment flasks (Corning). At day 1, 50% more mTeSR was added, and the final volume was supplemented with 10 μM SB431542 (Sigma) and 1 μM dorsomorphin (EMD Biosciences, catalog no. 171261). At day 3, medium was changed into 1:1 mTeSR and KOSR medium supplemented with 10 μM SB431542 and 1 μM dorsomorphin. KOSR medium consisted of 15% KOSR in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) with penicillin (10,000 U) and streptomycin (1 mg/ml) (PS; Gibco), l-glutamine, and nonessential amino acids (NEAAs; Invitrogen). The next day, the medium was changed into 1× KOSR medium supplemented with 10 μM SB431542 and 1 μM dorsomorphin. On day 5, the medium was switched to neural induction medium (NIM) supplemented with 10 μM SB431542, 1 μM dorsomorphin, ascorbic acid (0.2 μg/ml) (AA; Sigma), 1 μM retinoic acid (RA; Sigma), and 1 μM smoothened agonist 1.3 (SAG; EMD Biosciences). NIM consisted of DMEM/F12 with PS, l-glutamine, NEAAs, 0.16% d-(+)-glucose (Sigma), heparin sulfate (2 μg/ml), and 1% N2 supplement (Gibco). From day 7 to day 24, the medium was changed every other day with NIM supplemented with AA (0.2 μg/ml) (Sigma), 1 μM RA (Sigma), and 1 μM SAG (EMD Biosciences). At day 24, the embryoid bodies were dissociated, and GFP+ motor neurons were sorted using FACS (41, 42). FACS-purified motor neurons were plated onto a primary glia monolayer (42,000 glia cells per well) or Matrigel-coated poly-d-lysine/laminin–coated eight-well chamber slides (BD Biosciences) in equal numbers at a concentration of 20,000 to 25,000 or 40,000 cells per well, respectively. For the experiments shown in Fig. 1 (F and G), cells were plated at a concentration of 100,000 cells per well.

Glia cultures

Primary glia cultures were established as described by Di Giorgio et al. (18). When glia reached confluency, they were plated onto eight-well chamber slides (BD Biosciences).

Cocultures and treatments with chemical compounds

Post-treatment. The day after plating motor neurons, cocultures were treated with DMSO or compounds: BW A868C (Cayman Chemical), BW 245C (Cayman Chemical), or MK0524 (Santa Cruz Biotechnology) in motor neuron medium every other day. Motor neuron medium was used as described by Di Giorgio et al. (18). After 10 days of culture, cocultures were fixed and analyzed.

Pre-treatment. Confluent glia were pretreated for 1× 48 hours with DMSO or compounds in glia medium. Glia were washed before motor neurons were plated. After 10 additional days of culture, cocultures were fixed and analyzed.

Astrocyte coculture assays. Astrocytes were FACS-sorted for GFAP::GFP and then cultured for 72 hours in glia medium on polyorthinine-laminin–coated plates at a concentration of 40,000 cells per 384-well plate. Purified motor neurons were plated in motor neuron medium and cocultured for an additional 10 days in the presence of DMSO or compounds. Cocultures were fixed, stained for MAP2 and Hoechst, and analyzed.

Microglia coculture assays. Microglia were FACS-sorted for CD11b and then pretreated for 1× 48 hours, with DMSO or compounds, in glia medium. Microglia were washed twice and spiked onto 1-day-old culture of motor neurons at a concentration of 1000 microglia per well. Cocultures were cultured for an additional 10 days and fed with motor neuron medium supplemented with 50% conditioned medium (motor neuron medium conditioned for 2 days on nontransgenic glia). Cocultures were then fixed and analyzed.

Human astrocyte coculture. Astrocytes were obtained from ScienCell. Human astrocytes were cultured on poly-l-lysine–coated 24-well plates for 72 hours in human astrocytes medium (ScienCell) at a concentration of 42,000 cells per well. Then, purified motor neurons were plated (25,000 per well) onto astrocytes in motor neuron medium and cocultured for an additional 10 days in the presence of DMSO or compound. Cocultures were then fixed and analyzed.

Human microglia coculture. Microglia were obtained from 3H Biomedical. Human microglia were grown in poly-l-lysine–coated flasks in human microglia medium (3H Biomedical), and then cocultures were established and analyzed as described in “Microglia coculture assays.”

Human cell transduction. plv-AcGFP-SOD1 (wild type) and plv-AcGFP-SOD1G93A were provided by E. Fisher (plasmids 27138 and 27142, Addgene) (69). Transduced cells were then treated with DMSO or BW A868C for 48 hours and FACS-purified before analysis.

Immunocytochemistry

Samples were processed for immunocytochemistry as described in the Supplementary Materials. Primary antibodies used in this study were as follows: CD11b-647 (AbD Serotec), CD11b (Serotec Inc.), MAP2 (Abcam), CD68 (Thermo Fisher), Iba1 (Wako), GFAP (Santa Cruz Biotechnology), ChAT (Santa Cruz Biotechnology), S100b (Dako), and CD44 (eBioscience).

cAMP analysis. When glia reached confluency, they were pretreated with 3-isobutyl-1-methylxanthine at 500 nM for 30 min. Then, cells were treated with BW A868C (1 μM) or BW 245C (10 μM), collected at different time points, and analyzed as described in the cAMP EIA kit (Cayman Chemicals).

Real-time quantitative polymerase chain reaction

RNA was isolated with TRIzol (Invitrogen), treated with deoxyribonuclease (Invitrogen), and then transcribed into complementary DNA using iScript (Bio-Rad). Real-time quantitative polymerase chain reaction (PCR) was carried out with SYBR Green Quantitative RT-PCR Kit (Bio-Rad) and the iCycler system (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize all genes. Primer sequences are available upon request.

Animals and survival

B6SJL-Tg(SOD1-G93A)1Gur/J (The Jackson Laboratory) were bred with C57B6 DP1−/− mice (51). For purified astrocyte cultures, FVB/N-Tg(GFAPGFP)14Mes/J were crossed with B6SJL-Tg(SOD1-G93A)1Gur/J animals (The Jackson Laboratory).

RNA sequencing

RNA was harvested from at least two biological replicates using TRIzol (Invitrogen) according to the manufacturer’s directions. RNA quality was determined using BioAnalyzer (Agilent). RNA integrity numbers above 7.5 were deemed sufficiently high quality to proceed with library preparation. In brief, RNA-seq libraries were generated from 250 ng of total RNA using the Illumina TruSeq RNA kit v.2, according to the manufacturer’s directions. Libraries were sequenced at the Broad Institute’s Genomics Platform on a HiSeq 2500. RNA-seq reads were processed as previously described by Kiskinis et al. (70).

Statistical analysis

Student’s t test was used to test the significance between differences in mean values, and the null hypothesis was rejected at P < 0.05. Statistical analysis on animal survival and disease progression was performed using both one-way ANOVA and Mantel-Cox test.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/248/248ra104/DC1

Materials and Methods

Fig. S1. DP1 plays a role in glial toxicity to motor neurons.

Fig. S2. Characterization of DP1 mutant glia.

Fig. S3. Characterization of DP1 mutant compound animals.

Fig. S4. Characterization of inflammatory markers in DP1 mutant compound animals.

Fig. S5. M1 and M2 microglial activation.

Fig. S6. Human glial characterization.

REFERENCES AND NOTES

  1. Acknowledgments: We thank Eggan lab members especially J. Klim and N. Atwater. We thank P. Triphati and J. Zhou for help with purified astrocytes. Funding: This work was supported by grants from Howard Hughes Medical Institute (HHMI), New York Stem Cell Foundation, P2ALS, and Harvard Stem Cell Institute to K.E. A.S.d.B. was a Boehringer Ingelheim Fonds fellow. E.K. is a Charles King Trust Postdoctoral Fellow. N.S. was a 2011 Lilly Scientific Fellow and received grant from The Mochida Memorial Foundation for Medical and Pharmaceutical Research. B.N.D.-D. was an ALS Association Milton Safenowitz fellow. K.E. is an HHMI early career scientist. Author contributions: A.S.d.B. designed, performed, and analyzed experiments. K.K. assisted in all animal work and related experiments, including animal survival analysis, tissue harvesting, and processing. E.K. obtained the DP1 mutant animals and was involved in experimental design. N.S. cultured and characterized human fetal astrocytes. B.N.D.-D. performed RNA-seq analysis. K.E. conceived and supervised all experiments. Competing interests: A.S.d.B., K.K., and K.E. amended a patent application relating to DP1 as a therapeutic target of ALS. The other authors declare that they have no competing interests.
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