PERSPECTIVE Cell Cycle 14:23, 3679--3688; December 1, 2015; © 2015 Taylor & Francis Group, LLC

A perspective on stem cell modeling of amyotrophic lateral sclerosis A Sophie de Boer1,2 and Kevin Eggan1,* 1

The Howard Hughes Medical Institute; The Harvard Stem Cell Institute; The Stanley Center for Psychiatric Research; The Department of Stem Cell and Regenerative

Biology; Harvard University; Cambridge, MA USA; 2Department of Anatomy & Embryology; Leiden University Medical Center; The Netherlands

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Keywords: Amyotrophic Lateral Sclerosis, embryonic stem cells, induced pluripotent stem cells, motor neuron, glia, astrocyte, microglia Abbreviations: AA, anacardic acid; ADARB2, adenosine deaminase, RNA– specific, B2; ALS, Amyotrophic Lateral Sclerosis; ASO, antisense oligonucleotides; C9ORF72, chromosome 9 open reading frame 72; CRISPR, clustered regularly interspaced short palindromic repeats; DENN, neoplasma proteins (DENN); ER, endoplasmatic reticulum; ESCs, embryonic stem cells; FTD, fronto-temporal dementia; FUS, fused as sarcoma; GEFs, GDP/GTP exchange factors; hnRNPA1, heterogeneous nuclear ribonucleoprotein A1; iPSCs, induced pluripotent stem cells; OPTN, optineurin; PSCs, pluripotent stem cells; sALS, sporadic ALS; siRNA, small interfering RNA; SMA, spinal muscular atrophy; SOD1, superoxide dismutase 1; TDP43, TAR DNA binding protein 43; UBQLN, ubiquilin; UPR, unfolded protein response; VAPB/C, vesicle associated membrane protein-associated protein-B/C; VCP, valosin-containing protein. *Correspondence to: Kevin Eggan; Email: [email protected] Submitted: 08/31/2015 Revised: 09/08/2015 08/31/2015 Accepted: 09/07/2015 http://dx.doi.org/10.1080/15384101.2015.1093712 www.tandfonline.com

myotrophic lateral sclerosis is a complex neurodegenerative disease. Limitations in animal models have impeded progress in studying disease pathology and potential drug discovery. Here, we will review recent advances in the development of stem cell models for the study of ALS. Additionally, we will discuss the progress toward therapeutic development derived from these stem cell based assays.

Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that affects both upper and lower motor neurons in the brain and spinal cord. ALS is predominantly idiopathic; however genes involved in familial and potentially sporadic disease are being identified. Currently, mutations in SOD1 (superoxide dismutase 1), TDP43 (TAR DNA binding protein 43), FUS (fused as sarcoma), UBQLN, OPTN, VCP and C9ORF72 have been found to contribute to disease in patients.1-13 Progress in understanding disease mechanisms that underlie ALS has been slow. Consequently, therapeutic discovery has been hampered. Cell-types involved in disease processes are difficult to obtain and their only available source is post mortem patient tissue. Therefore, live culture of motor neurons directly from patients has been impossible. Even when it has been possible to obtain rare post mortem samples, opportunities to study neuronal pathology in a disease context have been limited, since at the time of post mortem tissue collection most motor neurons in the patients have degenerated. Mouse models that mimic disease progression and pathology have been successfully created for several mutations in ALS.14-16 However, they have limitations; treatments found to Cell Cycle

expand lifespan of these mice resulted in the conduction of more than 50 clinical trials in ALS patients. Yet, none of the trials resulting directly from these animal studies have had a successful outcome to date. In addition, mouse models only allow for highly penetrant mutations to be easily studied and thus cannot be used to examine sporadic disease, thereby excluding approximately 90% of all ALS patients.17 However, recent developments in pluripotent stem cell biology have provided opportunities to overcome these limitations. Stem cells enable disease modeling in a human cell context and provide the opportunity to study disease pathology of both familial and sporadic cases using patient derived cells. Pluripotent stem cells (PSCs) self renew and can therefore create an unlimited supply of daughter cells. Induced pluripotent stem cells (iPSCs), are PSCs that are generated from mouse or human somatic cells by the introduction of defined transcription factors that reprogramme the genome18 (Fig. 1A). iPSCs are similar to ESCs in developmental (pluri)potency, morphology, proliferation, ability to form teratomas in vivo, gene expression and DNA methylation patterns.19 Protocols to differentiate PSC into motor neurons are well-established.20,21 Presently, motor neurons can be derived rapidly and with high efficiency, by inducing neural patterning with several small molecules that modulate developmentally relevant pathways.20,21 Advances in the production of these motor neurons in large quantities have enabled analysis of previously intractable disease processes. This has led to better understanding of how and why motor neurons degenerate in diseases like ALS and could result in therapeutic opportunities. Initial ALS disease modeling focused on familial mutations, while more recent studies using 3679

lead to motor neuron degeneration in vitro and recapitulate previous findings in animal models in human cells. DiGiorgio et al.23 were the first to study effects of the SOD1G93A mutation in motor neurons derived from mouse ESCs.23 SOD1G93A mESCs were differentiated into motor neurons then survival and pathological hallmarks of disease were examined.23 SOD1G93A motor neurons had a lower rate of survival over time in culture. In addition to reduced survival, these vulnerable motor neurons displayed SOD1 protein inclusions in the cell body, perinuclear space and axons. SOD1G93A motor neurons also displayed increased levels of ubiquitin staining.23 Moreover, SOD1 mutant motor neurons expressed cytochrome-c and caspase-3, indicative of an activated apoptotic pathway.23 Overall, for the first time this study was able to recapitulate pathologies found in ALS patients and SOD1G93A mice in vitro using stem cell technologies. A more recent study by Yang et al. 24 took advantage of these SOD1 mutant mouse motor neurons to advance therapeutic development.24 Yang et al. 24performed a small-molecule screen to optimize survival in both wildtype and SOD1 mutant mESC-derived-motor neuFigure 1. Schematic overview of cell autonomous phenotypes found stem cell models of ALS (A) Schematic rons. Motor neurons were cultured overview of iPSC derivation. (B-E) Schematic overview of phenotypes found in SOD1, TDP-43, C9ORF72 and for 4 days in the presence of growth Sporadic iPSC derived motor neurons. factors. The growth factors were then withdrawn to induce cell iPSCs are enabling modeling of sporadic the Superoxide dismutase (SOD1) gene.1 death, and small molecules were added. disease as well. Mutations in this gene are found in ~20% Yang et al.24 found that when the small Here, we review recent attempts to of familial cases and are thought to act molecule Kenpaullone was added to the costudy ALS using stem cell models of dis- mainly through a toxic gain-of-function cultures, motor neuron survival improved.24 ease. Additionally, we discuss the use of mechanism.22 It has been found that over- They then discovered that Kenpaullone these assays for high-throughput screens expression of several SOD1 mutations can increased motor neuron survival by reducing with the ultimate goal of developing new lead to neurodegeneration in rodent mod- the levels of mutant SOD1 and ubiquitin as therapeutics. els. These animals recapitulate important measured by single cell imaging.24 It would hallmarks of ALS pathology in vivo, be of interest to further examine how Kenincluding mitochondrial dysfunction, paullone reduced SOD1 protein levels and Modeling cell-autonomous effects endoplasmatic reticulum stress and pro- how this in turn resulted in an increased surtein aggregation.1,16 However, recent vival of motor neurons. in vitro SOD1 More recent advances that enabled the advances in PSC biology and assays have One of the most common familial created new opportunities to study the generation of iPSCs from patients, have forms of ALS is the result of mutations in mechanisms by which SOD1 mutations led to an ever-increasing number of

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studies in human motor neurons (Fig. 1A). iPSC-derived motor neurons from patients carrying SOD1 mutations have abnormal cellular phenotypes, which were similar to those found in transgenic rodents with SOD1 mutations in vivo (Fig. 1B). Motor neurons carrying the SOD1 A4V mutation showed reduced survival compared with control motor neurons.25 These patient derived motor neurons exhibited several known phenotypic changes previously associated with ALS, which included neurite degeneration, reduced soma size and disturbed mitochondrial morphology.25 These findings were absent in control motor neurons in which this mutation was genetically corrected.25 Moreover, induction of the ER stress and the unfolded protein response (UPR) pathways was found in these SOD1 mutant motor neurons. Insoluble SOD1 protein accumulated upon proteasome inhibitor induced stress.25 Additionally, these SOD1 mutant motor neurons had more spontaneous electrophysiological activity compared to controls, an affect that could be reversed by treatment with retigabine, a kV7 channel activator.26 This hyperexcitability was similar to that previously reported in primary wildtype rat spinal cord slices which were treated with conditioned media derived from SOD1G93A primary murine glia.27 These rat neurons exhibited increased sodium intake and elevated firing patterns, which eventually led to motor neuron-specific death.27 In a separate study,28 iPSC-derived motor neurons harboring the SOD1 A4V and D90A mutations demonstrated similar alteration in dendrites as found by Kiskinis et al..25 Chen and colleagues 28 discovered that this shortened of neurite length was the result of neurofilament aggregation.28 The aggregation was motor neuron specific and was absent in control motor neurons. Over time in culture, neurofilament aggregation resulted in neurite swelling and eventually apoptosis, similar to neurite swelling previously observed in the SOD1G93A mouse model of ALS.28 Thus motor neurons derived from SOD1 mutant stem cells successfully recapitulated several phenotypes previously identified in SOD1 animal models and patients, including motor neuron

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degeneration. Additionally, new mechanisms of how mutant SOD1 induces these phenotypes were discovered. For instance, neurite swelling which was previously observed in SOD1G93A animal models was found to be the result of aggregation of neurofilaments.28,29 Nevertheless, more studies are necessary to find the underlying mechanisms of mutant SOD1 induced phenotypes and their contribution to disease as manifested in patients.

TDP43 Mutations in TAR DNA-binding protein 43 (TDP43) have been identified in ALS and fronto-temporal dementia (FTD).2,7,8,12,13 TDP43 is involved in various cellular processes, including transcription, RNA splicing and translational regulation. Over 50 point mutations in TDP43 have been found in ALS patients. The majority of these mutations are thought to affect RNA processing. Histological analysis in TDP43 patients post mortem tissues have shown mislocalization of TDP43 in the cytoplasm. This mislocalization has been identified in TDP43 cases as well as in several sporadic cases. However, it is unknown how these changes in localization result in motor neuron degeneration. Several advances using stem cell models have provided new insight into how mutations in TDP43 may contribute to neural degeneration. Similar to phenotypes found in SOD1 mutant motor neurons, TDP43 mutant motor neurons exhibited accelerated neuronal death and decreased neurite length30-32 (Fig. 1C). Unlike SOD1 mutant motor neurons, however, TDP43 mutant motor neurons had an increased abundance of insoluble TDP43 protein and impaired axonal transport of TDP43 mRNA.30-32 Additionally, TDP43 mutant motor neurons were more vulnerable to the stressor arsenite and had decreased expression of genes encoding cytoskeletal protein.30-32 Furthermore, increased expression of genes involved in RNA metabolism, including RNA processing, splicing and binding were found in TDP43 motor neurons. Cytoplasmic TDP43 aggregates were also observed in these TDP43 motor neurons.30-32 Treatment of TDP43 mutant

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motor neurons with anacardic acid (AA), a histone acetyltransferase inhibitor ameliorated several pathologies. AA treatment improved survival of TDP43 motor neurons after arsenite treatment. Additionally, AA treatment of TDP43 motor neurons resulted in a »150-fold decrease in TDP43 mRNA expression and a reduced level of TDP43 protein in the insoluble but not the soluble fraction. Further improvements upon AA treatment included increased neurite length. Additionally, AA treatment normalized the expression of several RNA metabolism-related genes, including genes involved in RNA binding, processing, splicing and transport in TDP43 motor neurons.30 Consistent with the TDP43 studies that showed abnormalities in motor neurons treated with the stressor arsenite,30,31 iPSC derived neurons carrying the TDP43 A90V mutation showed cellular abnormalities after treatment with the stressor staurosporine.3133 Abnormalities included TDP43 mislocalisation in the cytoplasm and decreased levels of TDP43.31-33 This decreased level of TDP43 resulted into dysregulation of miR9, both in TDP43 A90V and M337V mutant motor neurons.33 One of the shared pathologies found in several TDP43 mutant motor neurons was the mislocalization of TDP43. A recent study by Barmada et al. 34 found that improved nuclear localization increased survival of TDP43 M337V mutant motor neurons. This nuclear localization of TDP43 in motor neurons could be generated by autophagy induction with several compounds, including fluphenazine.34 Thus, autophagy induction could potentially become a new therapeutic avenue for patients carrying TDP43 mutations. Overall, stem cell models of TDP43 have provided insight into several disease pathologies, including TDP43 mislocalization, aggregation and impaired axonal transport (Fig. 1C). However, the exact molecular mechanisms by which TDP43 mutations cause motor neuron degeneration remain poorly understood.

C9ORF72 The repeat expansion in C9ORF72 is the most common mutation in ALS and FTD,

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found in 20-80% of familial, and 5-15% of sporadic cases.9,10 The recent identification of this form of familial ALS has resulted in the rapid development of various disease models designed to gain insight into disease pathogenesis. Several of these insights into how mutations in this relatively unstudied gene might result into motor neuron disease, have been developed through the use of iPSC-derived neurons from C9ORF72 patients. C9ORF72 neurons exhibited formation of RNA foci that co-localized with hnRNPA1 and Pur-a30 (Fig. 1D). Toxicity, by accumulation of cytotoxic poly-glycine– proline proteins was also observed in C9ORF72 neurons.35 Furthermore, these C9ORF72 neurons exhibited increased excitotoxicity when treated with glutamate, a phenotype that could be ameliorated both by inhibition of calcium channels and glutamate receptor inhibitors.36 Rescue of this exictotoxicity phenotype could be achieved by siRNA knockdown of ADARB2 RNA, a GGGGCCexp RNA binding protein.36 Knockdown of ADARB2 by siRNA reduced the formation of RNA foci by 49% and significantly reduced the susceptibility of C9ORF72 motor neurons to glutamateinduced excitotoxicity.36 C9ORF72 has been proposed to be a homolog of differentially expressed normal and neoplasma proteins (DENN).3739 This protein family consists of GDP/ GTP exchange factors (GEFs) that activate Rab-GTPases, which are involved in synaptic membrane trafficking. Consistent with this finding, isoforms A and B were detected in the membrane fraction of iPS derived C9ORF72 motor neurons.35 Another study reported motor neurons derived from iPSC carrying the repeat expansion showed increased nucleolar stress and increased sensitivity to proteotoxic stress, upon treatment with tunicamycin.40 Neurons derived from C9ORF72 patients also showed changes in excitability.26,35 Several genes involved in membrane excitability were differently expressed in C9ORF72 neurons compared to control, including KCNQ3 and DPP6.35 Interestingly, Wainger et al.26 recently reported an increase in total firing rate in these neurons, while Sareen et al.35 reported a decrease in excitability as measured by rheobase spiking rate.26,35 Differences reported might

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reflect an early hyperexcitable phase in susceptible neurons, which then die. Therefore, this hyperexictability might not be represented in the remaining neuron population. Hyperexcitability in one case could be reversed by treatment with retigabine, a kV7 channel activator.26 Moreover, treatments with antisense oligonucleotides (ASO) against C9ORF72 exon 2 and intron 1 reversed gene expression changes in genes affecting membrane excitability and reduced RNA foci formation, presenting exciting opportunities for therapeutic development.35 ASO treatment against exon 2 reduced all C9ORF72 isoforms and did not result in degeneration of motor neurons, thus pointing toward a gain of function phenotype.35 However, studies in C. Elegans and zebrafish found that reduced levels of C9ORF72 induced ALS pathology and both C9ORF72 post mortem neuronal samples and C9ORF72 iPSC derived neurons displayed reduced levels of C9ORF72 mRNA.36,41,42 Thus, additional study into C9ORF72 is warranted, in order to better understand the relative importance of gain and loss of function to overall patient phenotypes.

VAPB Mutations in vesicle associated membrane protein-associated protein-B/C (VAPB/C) have been identified in a small proportion of familial ALS and spinal muscular atrophy (SMA) cases.43 VAPB is expressed in the ER and pre-Golgi.44 Mutations in VAPB have been shown to affect the unfolded protein response and are thought to result in a loss of function, since reduced immunoexpression of VAPB is found in both the disease progression of SOD1 mutant mice and in human motor neurons in post-mortem spinal cord sections derived from sALS cases.45,46 Interestingly, fibroblasts, iPSC and iPSC-derived motor neurons derived from ALS8 patients carrying VAPB mutations, showed decreased expression of VAPB. This downregulation was similar to previous findings in mouse and human post mortem tissue derived motor neurons. Unfortunately, no change in protein unfolding was observed in the motor

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neurons derived from iPSC from ALS 8 patients.47 FUS Mutations in Fused in Sarcoma (FUS), a RNA binding protein involved in transcriptional regulation, are known to cause ALS.4,5 Relatively fewer studies have looked at FUS mutations in stem cell models. However, several disease mechanisms that were not previously described in mouse models of FUS have been identified using stem cells. Motor neurons derived from mESC transfected with FUS adenoviral vectors displayed cytoplasmic aggregate formation.48 Additionally, Wainger et al.26 found that motor neurons derived from iPSC of patients carrying mutations in FUS were hyperexcitable.26 Sporadic disease iPSC-derived motor neurons from known genetic cases have revealed several insights in ALS pathology as we have described above. However, another exciting opportunity resulting from the discovery of iPSCs is their ability to model sporadic disease in vitro for the first time. However, they do present some challenges. When we compare cells from sporadic cases to controls, changes we find may not be ALS related phenotypes but might merely be the result of distinct genetic backgrounds. As a result, disease phenotypes discovered using these cells could simply be due to different cell line variances. In familial cases, these problems can be overcome by using cells from controls that have been genetically “repaired” and thus isogenic, except for the disease causing mutation. However in sporadic disease this is impossible, due to the unknown genetic cause. It is therefore challenging to model sporadic cases, due to intrinsic variation and may require inclusion of many different patients for significance of any changes to be confirmed. Despite these challenges, iPSC derived motor neurons derived from sporadic ALS cases showed TDP43 pathology 49 (Fig. 1E). Spontaneous TDP43 aggregate formation was found in cell nuclei, mirroring aggregates found in the post mortem tissue of the donor from whom these

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iPSCs were derived.49 Furthermore, a small molecule screen using these motor neurons identified several previously FDA-approved compounds, including cardiac glycosides Digoxin, Lanatoside C and Proscillaridin A, offering an exiting opportunity for drug discovery and development through stem cell disease modeling.49 Stem cell models of ALS have validated several disease phenotypes in a human cell context, including protein aggregation and motor neuron degeneration. Additionally, new disease mechanisms have been discovered, that had not been observed in mouse models. These phenotypes could either be specific to humans or could be masked in animals. This masking could be due to redundant pathways, something which was previously observed using HPRT mutations in Lesch Nyhan Syndrome.50 Overall, while many distinct mutations cause ALS, thus far only a small number of shared pathogenic phenotypes have been discovered: motor neuron degeneration is usually preceded or accompanied by protein aggregation, mitochondrial dysfunction, ER stress, reduced neurite length, disturbed RNA processing and hyperexcitability. Furthermore, ALS mutant motor neurons are more susceptible to stressors. These shared pathogenic mechanisms are promising for potential drug development, as many different cases might benefit from a similar form of treatment. It is now pivotal to further examine the potential synergistic potential of these newly discovered candidate therapeutics for motor neurons. Since multiple pathogenic mechanisms are conserved among a variety of patients carrying different mutations, combinational therapy that affects several of these mechanisms simultaneously, might be more efficacious as treatment. Successes using these types of therapies have been accomplished in HIV patients. In these patients, the use of a combinational antiviral treatment resulted in greatly improved therapeutic responses. Exploring combinational treatments in animal and cell models for ALS would be of interest. A mixture of Retigabine, anacardic acid and autophagy stimulants would be one potential option to explore therapeutically using these models since

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this treatment would target hyperexcitability and RNA processing, processes that are both disrupted in various patients. However, particular patients might respond better to targeted therapeutic strategies against unique pathways, for example antisense oligonucleotide therapy for patients carrying the C9ORF72 repeat expansion. To identify the optimal treatment we would have to understand which pathogenic mechanisms are acting in a patient specifically, something iPSC derived motor neurons from a patient could provide.

Modeling non-cell autonomous effects in vitro While pathological events in motor neurons almost certainly play a role in disease there continues to be a growing interest in the role that other cells play in neural degeneration. Astrocytes, microglia and oligodendrocytes have been found to contribute to motor neuron degeneration in animal models of ALS. Some of these neighboring non-neuronal cells were identified as a participant in disease processes using a Cre/Lox strategy, which decreased mutant SOD1 expression in a specific cell-type.51-54 Reducing expression of mutant SOD1 (both G37R and G85R) in a subset of motor neurons (»20%), significantly delayed disease onset. Decreasing the levels of mutant SOD1 in either microglia or astrocytes also affected the course of disease. However, reduced levels of SOD1 in astrocytes and microglia slowed disease progression and did not change disease onset. In contrast, downregulation of mutant SOD1 in endothelial or muscle cells did not change the course of disease.51-56 While Schwann cells that had decreased expression of mutant SOD1 showed poorer survival in one study, a more recent report by Kang et al. 57 showed that depleting mutant SOD1 in PDGFRa+ oligodendrocytes improved disease outcome.57 Overall, multiple lines of evidence indicate that mutant SOD1 toxicity is not just in motor neurons. Astrocytes, microglia and oligodendrocytes have been shown to contribute to neurodegeneration and disease progression in SOD1 mutant animal models.

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Astrocytes Since several non-neuronal cells had been implicated in ALS pathology, assays have been developed to address whether these cells would also affect stem-cellderived motor neuron survival. DiGiorgio et al.,23,60 Nagai et al.59 and Marchetto et al. 58 used a co-culture of ESC derived motor neurons and SOD1 mutant glia to demonstrate this non-cell autonomous neurodegenerative effect in vitro.23,58-60 Glia harboring mutant SOD1 were toxic to motor neurons, while motor neurons cultured on wild-type glia did not show this toxicity (Fig. 2A). Furthermore, the toxic effect of glia was specific to motor neurons and was mediated through secreted diffusible factor(s) which induced inflammatory and apoptotic responses. Thus, similar to previous findings in rodents, stem cell derived motor neurons were susceptible to toxicity induced by non-neuronal glia cells. To begin to understand how mutant SOD1 glia affected motor neuron survival, gene expression of toxic mutant SOD1 glia was compared to that of supportive control glia. Glia expressing mutant forms of SOD1 showed upregulation of several inflammatory markers: Marchetto et al.58 reported increased expression of iNOS, chromagranin A, cystatin C and Nox2, while DiGiorgio et al.60 reported an increase in expression of the Prostaglandin D2 receptor.58,60 While the effects of fetal and neonatal derived SOD1 mutant astrocytes on motor neurons were analyzed by DiGiorgio et al.,23,58-60 Nagai et al.59 and Marchetto et al.,58 another study examined whether astrocytes derived from post-mortem samples would cause similar toxicity. Neural progenitors obtained from post-mortem samples of both sporadic as well as familial SOD1 patients induced degeneration of ESC derived motor neurons.61 As in the previous studies with fetal and neonatal astrocytes this effect was again motor neuron specific and mediated through secreted factors. Analysis of changes in these astrocytes demonstrated upregulation of inflammatory pathways in ALSderived astrocytes as well. NFkB and IFN-a signaling complexes were among the most highly expressed networks in these disease astrocytes.61 Overall, ALS

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of TDP43, decreased survival and increased levels of TDP43 65 (Fig. 2B). Overall, ALS astrocytes have consistently shown to effect motor neuron survival in culture, whether they were derived from SOD1, C9ORF72 or sporadic patients or animals. Therefore targeting astrocytes represents an important avenue for future therapeutic discovery. Microglia While originally non-cell autonomous stem cell based ALS studies mainly focused onto the effects of astrocytes, more recently interest has increased in studying the role of microglia in disease. Microglia are immune derived cells in the CNS, which upon activation release reactive oxygen species, pro-inflammatory cytokines, complement proteins, and neurotoxic molecules.66 Aberrant regulation of microglial activation can lead to neuronal dysfunction and death.66 Mutant SOD1 transgenic mice disFigure 2. Schematic overview of non-cell autonomous phenotypes found in stem cell models of ALS. (A-D) play microgliosis and inflammaSchematic overview of phenotypes found in SOD1, TDP-43, C9ORF72 and Sporadic astrocytes. tion accompanied by elevated levels of pro-inflammatory cytokines67-69 70,71 (Fig. 2A). It was mutant astrocytes negatively affect the sur- motor neuron toxicity from sALS astro- found that when SOD1 mutant microglia vival of stem-cell-derived motor neurons, cytes as was found previously in both were stimulated with LPS, they released which may be mediated through increased rodent and human ALS astrocytes.23,58- higher levels of TNF-a and Interleukin-6 neuroinflammation. However, further 60,62,63 It would therefore be interesting to compared with non-transgenic microglia. study to address if neuroinflammation determine whether the necroptosis path- Additionally, Xiao et al.72 discovered is the sole contributor of non-cell autono- way is affected in the rodent astrocytes that SOD1G93A mic-roglia released more mous induced motor neuron degeneration and neuroprogenitor derived astrocytes as nitric oxide, superoxide and less in ALS would be of interest. well, or if there are several mechanisms at IGF-1 than control microglia.72 When 62 More recently, Re et al. reported that play that are not shared between patient SOD1G93A mutant microglia were co-cultured with primary rat or human motor primary adult human astrocytes derived and rodent astrocytes. An additional study by Meyer neurons, a significant decrease in motor from both sporadic and familial cases could also induce motor neuron degenera- et al.,64 examined whether astrocytes neuron survival was observed, suggesting tion 62 (Fig. 2 A, D). However, interest- converted from fibroblast, from spo- that these changes are of direct relevance to ingly, motor neuron degeneration here radic, SOD1 and C9ORF72 patients, neuronal degeneration.63,72 Supporting the was not the result of increased astrocyte also induced toxicity (Fig. 2 A, C, D). notion that restoring normal microglial reactivity but rather that of the induction They discovered that indeed these astro- functionality can have beneficial effects in of the caspase-independent necroptosis cytes were toxic to mESC-derived motor ALS, introduction of normal microglia via pathway, which involves the protein neurons.64 bone marrow transplantation into kinase RIP1. Motor neuron survival could In contrast, iPSC derived astrocytes from SOD1G93A animals slowed disease probe rescued by inhibition of RIP1 by either patients harboring mutations in TDP43 did gression.73 In total, previous studies of necrostatin-1 or RIP-1 shRNA.62 Intrigu- not trigger toxicity when co-cultured with microglia in the ALS mouse model seem to ingly, Re et al. 62 did not find a neuroin- hESC-derived motor neurons.65 However, suggest that identifying factors capable of flammatory response underlying the these astrocytes did exhibit mislocalization either normalizing microglial function, or

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slowing their toxic activities could be of therapeutic benefit. Currently, stem cell models of disease are being employed to further explore the role of microglia in ALS. The first of these stem cell assays studying the effects of microglia in ALS was by Hoing et al.74 They developed a high-throughput screen analyzing microglia-induced toxicity in hESC-derived motor neurons.74 Microglia were activated using IFN gamma and LPS, treated with compounds and motor neuron survival was subsequently analyzed. Several hit compounds were identified, most of which played a role in stimulating Nrf2 target genes. These compounds increased the survival of hESC-derived motor neurons and mutant SOD1 astrocytes.74 This study specifically addressed microglia activation by IFN gamma and LPS and showed that ameliorating this activation improved motor neuron survival even on mutant SOD1 astrocytes. It demonstrated that reducing generic neuroinflammation might be a fruitful therapeutic strategy. It now remains to be addressed if this strategy will be effective in animal models as well. Another study by Frakes et al.75 did address if the effects of inhibiting microglia activation would extend survival of the SOD1G93A mice. They showed that reduced expression of NFkB prolonged disease progression and thereby lifespan in the SOD1G93A mouse model, by reducing microglia activation.75 In addition, they co-cultured adult microglia from these animals with mESC derived motor neurons. SOD1 mutant microglia were found to be toxic to motor neurons. However, this toxicity was eliminated when NFkB was inhibited. Furthermore, overexpression of the NFkB inhibitory protein IkBa in SOD1 mutant microglia also rescued motor neuron survival.75 In all, inhibition of NFkB is a very promising therapeutic strategy, which will hopefully soon be tested in a clinical setting. Another promising therapeutic target was found in our recent study. DiGiorgio et al.60 originally described the Prostaglandin D2 DP1 receptor as a therapeutic target in glia mediated toxicity. We recently validated this target in a transgenic animal models of ALS and identified that this protective

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effect was mediated by reduced microgliosis both in vivo as well as in vitro.60,63 Stem cell models of ALS have successfully recapitulated known phenotypes from patients.23,25,58-60 However, in vivo validation of a therapeutic target newly discovered in a stem cell disease model was lacking. Our recent study identified the DP1 receptor as a mediator of microglial toxicity to motor neurons in ALS. Furthermore, we validated for the first time a therapeutic target, DP1, discovered in a stem cell model of disease.63 Interestingly, previous studies have suggested that the DP1 receptor is necessary for mast cell maturation. Without the receptor, mast cells cannot participate in a normal inflammatory response.76 Additional experiments have also implicated DP1 in mast cell induced neuronal hyperexcitability.77,78 In these studies DP1 inhibition by BW A868C reduced the levels of extraneous firing when mast cells were co-cultured with primary neurons. Agonizing DP1 in mast cells using BW 245C caused these immune cells to induce an increase in neuronal excitability.77,78 This observation is of particular interest given that it has recently been shown that conditioned media from SOD1G93A glia can induce motor neuron hyperexcitability in rat spinal cord slices.27 Further studies of whether activated microglia share some properties with mast cells may be warranted. Microglia activation is an important hallmark of ALS. Several studies have already shown therapeutic benefit of inhibition of this activation. While its effects in patients still needs to be addressed, it does present exciting therapeutic targets. It remains of particular interest to examine if Nrf2, NFkB and DP1 inhibition act through a similar pathway or if a combined effect of inhibition of all 3 targets would result in an even longer lifespan in animals. Limitations There remain several limitations in the use of stem cells assays of ALS that still need to be addressed for these models to reach full potential. While differentiation protocols for several disease-implicated cell types have been successfully established, including motor neurons and

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astrocytes, others such as microglia have not. Additionally, oligodendrocytes have been recently identified to play an important role in the non-cell autonomous effects in ALS; to further explore their contribution to disease, protocols to derive oligodendrocytes from stem cells need to be established. Further optimization of protocols for cortical motor neurons, upper motor neurons, which have also shown to be affected during disease as well as in culture, is necessary.79 Furthermore, differentiation protocols for related yet disease-resistant cell-types such as oculo motor neurons would greatly advance the field. However, even optimized protocols have their limitations. Different cell lines and even different passages of a single cell line have different potentials to develop into specific cell-types, underscoring the dire need for purified populations of target cells, either by the development of reporter cell-lines, the use of cell surface markers or by virus transduction with a reporter construct. However, pure populations of cells derived from various cell lines can still greatly vary in behavior due to changes in their genetic background. Therefore, in a worst-case scenario, disease phenotypes discovered using these cells could merely be the result of cell line differences. Thus, idiopathic cases should be ideally modeled using purified target cell types from controls in which only the disease causing mutation has been corrected. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR) technology enables efficient and low-cost genomic editing of cell lines to either correct or introduce a disease causing mutation. While several of the above described studies, including Donnelly et al.,36 Kiskinis et al.25 and Wainger et al.,26 employed these techniques at least in part, others did not.26,36,80 A more routine use of these genetic correction techniques in combination with a purified target cell population would likely result in the discovery of more subtle and robust phenotypes and will thereby avoid misinterpretation. Another challenge in using stem cells to model ALS is the relatively immature status of the cell types used to investigate this late-onset disease. While advances have been made to optimize differentiation

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protocols and maturation of motor neurons and astrocytes in vitro, most phenotypes were only discovered after addition of stressors to the culture. It remains unclear if these hallmarks of disease are only the result of environmental stress, a culture artifact, or if fully mature cells would eventually develop these phenotypes over time. Moreover, a complex combination of cross talk between different cell-types might be necessary to provide disease pathophysiology. Therefore, an effort should be made to further develop these more complex co-culture systems. Future perspectives The use of stem cells, which can potentially be differentiated into any cell type and maintained indefinitely, holds great promise for further discovery of disease mechanisms, especially in otherwise difficult-to-obtain human cells. Additional advantages could be the discovery of novel therapeutics using high throughput screening using small molecule libraries and the opportunity for personalized screening of these new therapeutics or validation of the currently only FDA approved therapeutic Riluzole.81 More broadly, general drug safety testing could become standard in highly affected cell types, e.g. cardiomyocytes, improving general drug safety and accelerating clinical trials. Widespread use of stem cells to model ALS could result in faster development and approval of much needed therapeutics. Recent therapeutic discoveries found using stem cell models of ALS will need further exploration: will these targets work synergistically or are they part of a shared pathway? Additionally, most targets were found using SOD1 models so it remains an open question whether they will have therapeutic effects on models that carry a different mutation. Retigabine acts on motor neurons specifically to reduce hyperexcitability and would therefore be unlikely to share overlap with the targets found to act in glia cells: Nrf2, NFkB and DP1. However, since Nrf2, NFkB and DP1 are all inflammatory targets that act on microglia, their epistatic interactions need to be evaluated to come to a conclusion. Nrf2 has been

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shown to directly cross talk with NFkB during inflammation, they antagonize each other in the RAC1-inflammatory pathway. Moreover, while currently no direct interaction between these inflammatory targets and DP1 have been reported, Nrf2 has been shown to be a downstream target of 15-deoxy-Delta (12,14)-prostaglandin (2), a degradation product of Prostaglandin D2. It will therefore be important to assess if combined targeting of these targets will result in an additional increase in survival in animals and stem cell models, or if no synergistic effect is observed. Additionally, the feasibility to drug these targets in a combinational therapy needs to be examined, together with the ability of these compounds to cross the blood brain barrier. Several specific DP1 antagonists have been recently identified. Safety of 2 compounds antagonizing DP1, AMG 853 and Laropripant have been assessed in clinical trials. While treatment with AMG 853 resulted in no serious side effects, a longterm combinational therapy of laropripant and niacin, a cholesterol-lowering drug, did result in side effects associated with skin, gastrointestinal and musculoskeletal system. However, whether these side effects were the result of the DP1 antagonist or niacin remains unclear. When Laropripant was tested in the absence of niacin in asthma patients, a safety profile comparable to the placebo group was detected, offering a promising outlook on the safety of drugs targeting this receptor. While several Nrf2 activating compounds have been developed, CXA-10 is, to the best of our knowledge, the only one being currently tested for safety in a Phase I clinical trial. Additionally, over 800 compounds have been shown to have antagonizing effects on NFkB, some which have successfully been tested for safety with no adverse effects, including, a-lipoic acid and curcumin, while others did induce some side effects, including pitavastatin. It now remains pivotal to address the ability of these drugs to cross the blood brain barrier as well as address the options and potential of a combinational therapy. Overall, stem cell models of ALS have shown successful recapitulation of known phenotypes from patients as well as

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revealing new mechanistic insights. It remains pivotal to look for these pathological phenotypes in patients. Large cohorts of patients, having similar as well as different genetic backgrounds as the cells they were discovered in, are necessary to validate these pathological phenotypes as well as newly discovered therapeutics. Disclosure of Potential Conflicts of Interest

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Acknowledgments

Kevin Eggan is a HHMI early career scientist. Kevin Eggan and A. Sophie de Boer also gratefully acknowledge support from Project ALS and Target ALS. Funding

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Volume 14 Issue 23