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. Author manuscript; available in PMC: 2014 Jun 6.
Published in final edited form as: Cell Stem Cell. 2013 Apr 18;12(6):713–726. doi: 10.1016/j.stem.2013.04.003

A Small Molecule Screen in Stem Cell-derived Motor Neurons Identifies a Kinase Inhibitor as a Candidate Therapeutic for ALS

Yin M Yang 1,2, Shailesh K Gupta 1,2,*, Kevin J Kim 1,2,*, Berit E Powers 1,2,*, Antonio Cerqueira 1,2, Brian J Wainger 5,6, Hien D Ngo 1,2,3, Kathryn A Rosowski 1,2,4, Pamela A Schein 1,2, Courtney A Ackeifi 1,2, Anthony C Arvanites 2, Lance S Davidow 2, Clifford J Woolf 5, Lee L Rubin 1,2
PMCID: PMC3707511  NIHMSID: NIHMS465742  PMID: 23602540

Abstract

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease, characterized by motor neuron (MN) death, for which there are no truly effective treatments. Here, we describe a new small molecule survival screen carried out using MNs from both wildtype and mutant SOD1 mouse embryonic stem cells. Among the hits we found, kenpaullone had a particularly impressive ability to prolong the healthy survival of both types of MNs that can be attributed to its dual inhibition of GSK3 and HGK kinases. Furthermore, kenpaullone also strongly improved the survival of human MNs derived from ALS patient induced pluripotent stem cells and was more active than either of two compounds, olesoxime and dexpramipexole, that recently failed in ALS clinical trials. Our studies demonstrate the value of a stem cell approach to drug discovery and point to a new paradigm for identification and preclinical testing of future ALS therapeutics.

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset disease that causes degeneration of both upper and lower motor neurons (MNs). ALS generally progresses rapidly, with a mean survival time of between three and five years. Approximately 10% of cases are familial (inherited), and 20% of these are caused by mutations in the SOD1 gene (Brown, 1997; Boillée et al., 2006). Although SOD1-associated ALS accounts for only ~2% of all cases, it has been the most studied form due to the early discovery of disease-causing mutations and the availability of mouse models. Mutations in the SOD1 gene cause a gain of toxic, rather than loss of normal, function. Exactly how this causes MN death is still unclear, but it is now well accepted that cell autonomous and non-cell autonomous mechanisms can contribute to degeneration (Di Giorgio et al., 2007; Nagai et al., 2007; Ilieva et al., 2009). A more recent breakthrough has identified TAR-DNA binding protein-43 (TDP-43) as a major component of protein aggregates found in sporadic ALS and non-SOD1 familial ALS cases (Arai et al., 2006; Neumann et al., 2006). Mutations in TARDBP, the gene encoding TDP-43, were found in ~4% of familial ALS cases (Van Deerlin et al., 2008). Further, a hexanucleotide repeat expansion within the C9orf72 gene has recently been identified as the most frequent pathogenic cause of ALS thus far, accounting for about 6% of sporadic ALS cases, and 36-40% of familial ALS cases in Europe and the USA (Renton et al., 2011; Majounie et al., 2012). Thus, there may be numerous pathogenic initiators of ALS, including oxidative stress, protein misfolding and aggregation, RNA processing, mitochondrial dysfunction, excitotoxicity, neuroinflammation, axonal transport defects, and neurotrophin depletion (Joyce et al., 2011).

Riluzole is currently the only approved treatment for ALS. It is thought to act by reducing an excitotoxic component of the disease, but prolongs life by only 2 to 3 months and provides little functional improvement (Miller et al., 2007). While there has been a great deal of effort to discover better treatments, both olesoxime and dexpramipexole (Cudkowicz et al., 2011; Sunyach et al., 2012), two promising compounds, failed in phase III ALS trials just in the past year. Both had been tested in a mouse model of ALS, but neither had been tested on rodent ALS MNs. Needless to say, neither had been tested on any type of human MN in vitro either. Thus, it may be that having better methods for identifying promising drug candidates for ALS and rigorously evaluate them in vitro prior to testing them in the clinic would greatly improve the chances of discovering better treatments for this disease.

In the last few years, simple protocols for differentiating mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into MNs have become available (Wichterle et al., 2002; Miles et al., 2004; Boulting et al., 2011). For the present study, we generated large numbers of MNs from wild-type mouse ESCs and from mouse ESCs carrying a human SOD1G93A transgene (Di Giorgio et al., 2007) and used both in trophic factor withdrawal screens to identify small molecules that promote MN survival. Of the compounds that increased survival of both cell types, the multi-kinase inhibitor, kenpaullone, stood out. A variety of experiments showed that kenpaullone not only keeps MNs alive for several weeks in the absence of added trophic support, but maintains neuritic processes, synapses and normal electrophysiological characteristics. We show that kenpaullone’s potent effects are mediated via dual inhibition of GSK-3α/β and HGK (MAP4K4), a kinase upstream in the phospho-c-jun mediated neuronal apoptosis pathway (Yao et al., 1999). Furthermore, kenpaullone was also able to improve survival of MNs produced from wildtype and two different types of ALS patient-derived iPSCs, while olesoxime and dexpramipexole were less effective. In that regard, although kenpaullone was simply a hit compound from our screen and not chemically optimized in any way, it was superior to two compounds recently tested in the clinic.

Results

Assay development for motor neuron survival screens

We produced MNs from both wild-type (Hb9::GFP) and SOD1G93A (SOD1G93A/HB9::GFP) mouse ESCs using modifications of standard protocols (Figure 1A and Experimental Procedures) and were able to generate cultures with 30%~50% MNs. As reported by Di Giorgio et al. (Di Giorgio et al., 2007), we found that there was no significant difference in basal survival between HB9::GFP and SOD1G93A/HB9::GFP cultures in the first week, although survival of SOD1G93A/HB9::GFP MNs was somewhat more variable. We chose a standard model of inducing death – trophic factor withdrawal – and applied it to both types of MNs. Various plating densities and times for initiation and duration of trophic factor withdrawal were optimized for a 384-well HTS format, so that we achieved ~80% MN death in 3 days (Figure S1). Cycloheximide (CHX), a protein synthesis inhibitor that blocks apoptosis (Mattson and Furukawa, 1997), was identified as a hit in a trial screen (Figure S1C) and was used as a positive control compound.

Figure 1. see also Figure S1 and Table S1. Identification of small molecules that promote the survival of ESC-derived MNs.

Figure 1

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(A) Overview of directed differentiation and MN screening flow. Ag, Shh agonist; RA, retinoic acid. Scale bar represents 200 m.

(B) Plate layout in the form of a heat map of a 384-well screening plate. Positive controls are in left top (–TF/+cycloheximide) and right (+TF) columns and negative controls (−TF, DMSO-treated) are in left bottom. Representative images of HB9::GFP MNs are shown for controls and for a hit well in which cells were treated with kenpaullone. Each image shown represents a field taken within an individual well (approximately one twelfth of the well). Scale bar represents 200 m.

(C) Scatter plots showing screening results (points in red correspond to individual library compounds).

(D) Dose curves of a compound that scored only in HB9::GFP MNs (Tyrphostin A9), a compound that scored only in SOD1G93A/HB9::GFP MNs (MDL 28170), and two compounds that scored in both HB9::GFP and SOD1G93A/HB9::GFP MNs (kenpaullone and CP55940). Data are presented as mean ± SD. Note that in both HB9::GFP and SOD1G93A/HB9::GFP screens, average MN survival in negative control wells (−TF, DMSO-treated) was about 30% of that in +TF control wells.

(E) Image-based analyses allowed for excluding putative hit compounds that did not preserve MN integrity. Images shown here are (left) of a compound that preserved normal cell morphology including an intact neurite network (arrows: examples of visible neuronal processes) and (right) of a compound that resulted in an increased number of MNs, but mostly with small soma and few neurites. Scale bar represents 100 m.

Identification of compounds that promote MN survival

A collection of approximately 5000 small molecule compounds including known drugs, bioactives and other annotated chemicals was screened at three concentrations (Figure 1B,C). Hits were defined as compounds that both robustly increased the number of surviving MNs compared to wells without trophic factors and preserved cell morphology (size of soma, presence of neurites; Figure 1E). Hit compounds from the primary screen were retested in dose-response survival assays and then categorized by annotated activities. A significant number of hits or hit categories for HB9::GFP or SOD1G93A/HB9::GFP MNs act on targets previously shown to be neuroprotective or involved in ALS. Nine compounds that inhibit protein or DNA synthesis improved survival of one or both types of MNs. These compounds are well known to be anti-apoptotic (Mattson and Furukawa, 1997; Martin, 2001). The matrix metalloproteinase (MMP) inhibitor, 1,10-phenanthroline monohydrate, promoted the survival of both types of MNs. MMPs have been implicated in MN degeneration in ALS, and MMP inhibitors have been shown to extend the survival of ALS mice (Lorenzl et al., 2006; Niebroj-Dobosz et al., 2010). CP55940 (a cannabinoid receptor agonist) promoted the survival of both MN types (Figure 1D). Cannabanoid receptors have previously been shown to be elevated in spinal cords of symptomatic SOD1G93A mice, and treatment with cannabinoid receptor agonists delayed disease onset and prolonged survival (Kim et al., 2006; Shoemaker et al., 2007). Additionally, the calpain inhibitor, MDL 28170, promoted survival of SOD1G93A/HB9::GFP MNs (Figure 1D), consistent with studies in which calpain inhibition prolonged the viability of SOD1G93A MNs in culture and the lifespan of SOD1G93A mice (Wootz et al., 2006; Tradewell and Durham, 2010). Ligands for neurotransmitter receptors (e.g. A 77636 hydrochloride and 3-Tropanylindole-3-carboxylate methiodide), and compounds targeting calcium channels (e.g. the calcium agonist FPL-64176) also scored as hits in one or both types of MNs. This came as no surprise as neuronal activity and calcium flux are crucial regulators for neuronal survival, and ALS is known to involve the dysregulation of both (Sandyk, 2006; Grosskreutz et al., 2010). Finally, hit compounds included several kinase inhibitors. Tyrphostin A9 (a multi-kinase inhibitor) promoted survival of HB9::GFP MNs alone, while Kenpaullone (annotated as a GSK-3 inhibitor) strongly increased survival of both MN types (Figure 1D). We chose to pursue compounds that affected both MN types reproducibly, since they might have the best chance of working broadly across different ALS disease types (These hits are listed in Table S1).

Kenpaullone and some other GSK-3 inhibitors promote survival

In follow-up studies, we focused on kenpaullone, the compound that consistently rescued survival most effectively for wild-type and mutant MNs. Kenpaullone reportedly acts on a biologically meaningful and pharmacologically “druggable” target, GSK-3. We therefore tested additional commercially available GSK-3 inhibitors in our survival assay. Several did promote survival, but their effects were often weaker and more variable than those of kenpaullone. Surprisingly, alsterpaullone, a close structural relative of kenpaullone, was less consistent and was often toxic at higher doses (Figure S2A). GSK-3 inhibitor VIII (Anastassiadis et al., 2011) and GSK-3 inhibitor XIII, showed consistent, albeit significantly diminished, survival activity (Figures S2A). Still others, such as CHIR99021 and CHIR98014, thought to be among the most specific inhibitors (Bain et al., 2007), did not promote survival at all (Figure 2A). These experiments suggested that while GSK-3 inhibition might play a role in promoting survival, the particularly pronounced effect of kenpaullone could have resulted from additional molecular activities.

Figure 2. see also Figure S2. Kenpaullone promotes MN survival through a cell autonomous mechanism.

Figure 2

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(A) Survival effects of kenpaullone and more specific GSK-3 inhibitors, CHIR 98014 and CHIR 99021. Kenpaullone clearly has particularly strong activity. In this experiment, the MN survival in –TF/DMSO-treated controls was 18% (for HB9::GFP) and 35% (for SOD1G93A/HB9::GFP) of that in their respective +TF controls.

(B) Reducing the number of progenitor cells using Ara-C does not diminish the effectiveness of kenpaullone. Olig2 staining was used to identify MN progenitors. Ara-C treatment (between days 2 and 4) significantly reduced the number of Olig2+ cells (p<0.01). At the same time, kenpaullone’s effect was not reduced by Ara-C treatment; in fact, there was a small but statistically significant (two-way ANOVA, p=0.0002) increase in MN survival by kenpaullone after Ara-C treatment. Scale bar represents 200 m.

(C) The effect of kenpaullone remains essentially unchanged in highly enriched Hb9::GFP MN cultures. FACS purified MNs were treated with Ara-C to further ensure that any residual progenitor cell proliferation would be minimized. Images show that these cultures had very few non-MNs. In spite of that, kenpaullone was able to improve MN survival to a comparable degree to that observed in mixed cultures. Note that the MN survival in –TF control was 38% of that in +TF control in this experiment. Scale bar represents 100 m.

Data in this figure are presented as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 (Student’s t test, two-tailed, compared to –TF controls).

Kenpaullone increases survival, but not the production, of new MNs

Recent publications suggest that GSK-3 may be involved in regulating the proliferation and differentiation of neural progenitors (Hur and Zhou, 2010; Lange et al., 2010). To confirm that the increase in MN numbers produced by kenpaullone treatment resulted from increased survival of existing MNs, we treated cultures with the antimitotic agent Ara-C to eliminate dividing progenitors before trophic factor withdrawal and treatment (Figure 2B). While treatment with Ara-C significantly reduced the number of Olig2+ progenitor cells, it was in fact associated with a small increase in the effect of kenpaullone. We also used BrdU to label proliferating cells and any MNs derived from them (Figures S2B and S2C). We did not find any BrdU/GFP double-positive cells. These studies strengthen the conclusion that kenpaullone has a genuine survival-promoting effect.

The effect of kenpaullone is cell autonomous

ESC-derived MN cultures are heterogeneous, containing 50% to 70% non-neuronal cells or other types of neurons. To determine if kenpaullone exerts its protective effect in a cell autonomous or non-cell autonomous way, we used FACS to purify MNs based on their Hb9::GFP expression and established highly enriched cultures (Figure 2C). We then treated them with Ara-C to further limit the number of proliferating progenitors. When trophic factors were removed from these cultures, kenpaullone treatment still promoted survival, with a dose curve similar to that in mixed cultures. Thus, kenpaullone seems to act on MNs themselves.

Kenpaullone also promotes survival when death is initiated by other types of stimuli

The PI3K/Akt pathway is well known to regulate neuronal survival including that of MNs (Brunet et al., 2001; Barthélémy et al., 2004). Furthermore, PI3K/Akt activity is decreased in MNs of both sporadic and familial ALS patients, as well as in mutant SOD1 mice (Koh et al., 2004; Dewil et al., 2007). We sought to examine if kenpaullone or a more specific GSK-3 inhibitor (Inhibitor VIII) could protect MNs from death caused by inactivation of PI3K/AKT. The PI3K inhibitor LY294002 induced dose-dependent death in both HB9::GFP and SOD1G93A/HB9::GFP MNs (Figure 3A). However, when kenpaullone was co-incubated with LY294002, MN death was significantly attenuated. In contrast, although Inhibitor VIII was able to protect slightly after trophic factor withdrawal, in this case, it produced only a very small increase in HB9::GFP MN survival and did not have any noticeable effect on SOD1G93A/HB9::GFP MNs. We also tested PI-103, a more potent and selective PI3K inhibitor, with similar results (Figure 3B). Thus, kenpaullone’s strong effects are not restricted to protection after trophic factor withdrawal.

Figure 3. see also Figure S3. Kenpaullone treatment improves survival when MN death is initiated by other perturbations.

Figure 3

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(A) and (B) Effects of kenpaullone and GSK-3 inhibitor VIII on MN death induced by PI3K inhibitors. LY294002 (A) or PI-103 (B) was added to cultures on day 4, with or without concurrent treatment with 10 M kenpaullone or 10 M GSK3 inhibitor VIII. Cells were fixed on day 7 for survival analysis. Survival is measured with respect to cells treated with DMSO alone. * p<0.05, ** p<0.01, *** p<0.001 (Student’s t test, two-tailed, compared to 0 M LY294002 or PI-103).

(C) Kenpaullone also blocks death of MNs accelerated by co-culture with SOD1G93A astrocytes. Hb9::GFP MNs were FACS sorted and plated on a confluent monolayer of wild-type or SOD1G93A astrocytes. Kenpaullone was added to cultures 24 hrs later and was refreshed every three days by replacing half of the old medium with fresh medium containing 1X concentration of the compound. Cells were fixed after nine days of compound treatment and MN survival was determined. Trophic factors were present throughout the experiment. Data are presented as percent survival normalized to that of DMSO treated MNs plated on wild-type astrocytes. Again, kenpaullone was effective in decreasing the extent of death.

Data are presented as mean ± SEM.

Several studies have shown that astrocytes derived from ALS patients or animal models, especially mutant SOD1-bearing astrocytes, contribute to MN death (Di Giorgio et al., 2007; 2008; Marchetto et al., 2008; Díaz-Amarilla et al., 2011; Haidet-Phillips et al., 2011). We also observed that HB9::GFP MNs plated on a monolayer of SOD1G93A astrocytes survived less well than the same cells plated on wild-type astrocytes, with the effect becoming most obvious at later time points (Figure 3C). We found that kenpaullone improved MN survival on both wild-type and SOD1G93A astrocytes. In fact, kenpaullone treatment allowed MNs to survive as well on SOD1G93A astrocytes as on wild-type astrocytes. This result demonstrates that kenpaullone is able to act even when death is triggered by “toxic” glial factors.

Kenpaullone promotes long-term survival of wild type and mutant MNs in the presence or absence of trophic factors

We analyzed whether kenpaullone was able to promote survival in a trophic factor deficient environment for extended periods of time (Figure S3A). At all time points examined, kenpaullone improved survival of both HB9::GFP and SOD1G93A/HB9::GFP MNs, but the effect was proportionally greater in SOD1G93A/HB9::GFP cultures at the later time points perhaps because their survival was additionally compromised. In addition, we found that the basal level of cell death could be prevented by kenpaullone treatment even when cells were kept in neurotrophin-containing medium (Figure S3B). Under those conditions, as previously reported (Di Giorgio et al., 2007), the amount of death in SOD1G93A/HB9::GFP cultures began to exceed that in HB9::GFP cultures after 2 weeks. As the relative death of SOD1G93A/HB9::GFP MNs increased at those later times, the effect of kenpaullone again became relatively greater. By day 28, there was an 8-fold increase in the number of SOD1G93A/HB9::GFP MNs in medium with kenpaullone compared to medium without, compared to a 2.9-fold enhanced survival of wild type MNs (Figure S3C).

Effect of kenpaullone on MN morphology and function

To investigate whether MNs kept alive by kenpaullone in the absence of neurotrophic factors are truly intact, we evaluated a variety of morphological and electrophysiological properties of the surviving cells. First, we found that kenpaullone supported the structural integrity of MN processes even at extended time points (Figures S4A-S4F). We also used an automated imager to count the number of synapses per MN (defined as overlapping regions positive for the presynaptic marker synapsin and the postsynaptic marker PSD95 on individual MNs; Figure 4A). Again, kenpaullone was able to preserve morphological synapses on both MN types.

Figure 4. see also Figure S4. Kenpaullone preserves morphological and functional integrity of MNs even after long treatment periods.

Figure 4

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(A) Kenpaullone (5 M) increases the number of synapses per MN, even when analyzed on day 21 (after 17 days of kenpaullone treatment). Images show synapsin (yellow) and PSD95 (red) staining on a kenpaullone-treated MN. Arrows indicate examples of synapses on MNs. Data are presented as mean ± SEM. * p<0.05 (Student’s t test, two-tailed). Scale bar represents 50 m.

(B) Kenpaullone-treated MNs are electrophysiologically similar to those maintained in TFs. HB9::GFP MNs treated for 3 days in the absence of trophic factors (−TF/+kenpaullone) were compared to MNs that were grown in complete medium (+TF) in terms of functional sodium and potassium currents [1], action potentials [2], response to excitatory neurotransmitter (kainate, 100 M) [3], and response to inhibitory neurotransmitter (GABA, 100 M) [4].

Next, we compared the electrophysiological properties of MNs grown in complete medium with trophic factors (+TF) with those of cells in trophic factor-deficient (−TF) medium, with or without kenpaullone supplementation (Figures 4B and S4G). Attempts to collect data from cells grown in – TF medium without kenpaullone failed to obtain stable recordings despite a significant number of patch attempts. However, when we carried out whole-cell voltage-clamp recordings from MNs grown in +TF medium or –TF medium with kenpaullone, we found no significant differences in their properties (Figure S4G). In current clamp mode, depolarizing stimuli elicited robust action potentials in both groups. Two key measurements that reflect neuronal health are the resting membrane potential and input resistance, and we found that neither varied between the two groups (Figure S4G). We also recorded responses to excitatory neurotransmitters, such as kainate, and inhibitory transmitters, such as GABA. Again, we found no significant differences between groups. Together, these data show that kenpaullone treatment preserves neuronal health when trophic factors are removed.

Kenpaullone decreases mutant SOD1 levels

Since enhanced death undergone by SOD1G93A/HB9::GFP MNs, especially at later times, seems likely to relate to their expression of high levels of SOD1G93A protein, one possibility is that kenpaullone could act in part by reducing those levels. When we added kenpaullone to cultures on day 4 and analyzed them by Western blot on day 21, we found that SOD1G93A levels were sharply decreased both in the absence (Figure 5A) and the presence (Figure 5B) of trophic factors, Endogenous mouse SOD1 protein was not affected (data not shown), nor was there an effect on wild-type human SOD1 levels in cultures produced from an ESC line that overexpresses wild-type human SOD1 (wtSOD1/Hb9::GFP) (Figures 5A and 5B). Furthermore, single cell confocal imaging revealed that SOD1G93A levels were reduced in GFP+ MNs, both in the presence and absence of trophic factors (Figure 5C).

Figure 5. see also Figure S5. Kenpaullone treatment decreases mutant SOD1 protein levels.

Figure 5

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(A) In the absence and (B) in the presence of trophic factors, SOD1G93A protein, but not wt SOD1 protein, was decreased when cultures were treated with kenpaullone and analyzed on day 21. The antibody used was specific for human SOD1, as is seen by the lack of staining on wild-type mouse MNs (HB9::GFP).

(C) To confirm that the level of SOD1G93A protein was also decreased specifically in MNs, cultures were stained with an anti-human SOD1 antibody and analyzed by single cell imaging of GFP+ cells. By this analysis, again on day 21, the average amount of SOD1 per MN was decreased by kenpaullone treatment in both +TF and –TF conditions. Insets show higher magnification of cells contained within white circles. Scale bar represents 50 m.

Data are presented as mean ± SEM. p values were calculated with a two-tailed Student’s t test. n.s., not significant.

It has been shown that SOD1G93A begins to accumulate in ubiquitinated aggregates after 2 weeks of culture in neurotrophin-containing medium. This is approximately the time at which the rate of death of the mutant MNs, relative to that of wildtype MNs, begins to increase (Di Giorgio et al., 2007). We confirmed the appearance of aggregates in our cultures at those later time points (Figure 5C). We found no differences in mutant protein levels between untreated and kenpaullone treated cultures until at least 15 days of treatment (Figure S5A). We also measured MN levels of ubiquitin by single cell imaging and found it was reduced at days 21 and 28, but not at day 15, regardless of the presence or absence of trophic factors (Figure S5B). Thus, in addition to its early survival promoting activity, kenpaullone has a delayed effect on the accumulation of both SOD1G93A and ubiquitin, decreasing both at around the time at which proteins begin to aggregate and mutant MNs have an accelerated rate of death.

Mechanistic studies of kenpaullone’s activities

Kenpaullone is known to inhibit GSK-3, so we first sought to determine the role of GSK-3 in MN survival in our assays. To eliminate the possibility of functional redundancy, we employed a lentiviral-mediated shRNA knockdown strategy to reduce expression of both α and β isoforms of the enzyme (Figures S6A and S6B). In cultures infected with lentivirus carrying a non-silencing shRNA, less than 20% of MNs survived trophic factor withdrawal (Figure S6B). In contrast, in cultures receiving GSK-3 shRNAs, more than 50% of MNs survived. This survival was significant, but still substantially lower than that achieved with kenpaullone.

Since it was possible that we were unable to achieve higher levels of survival using shRNAs because of incomplete knockdowns, we used another approach to address the role of GSK-3 in survival after trophic factor removal. We produced MNs from the following mouse ESC lines: GSK3α−/− (GSK3α-KO), GSK3β−/− (GSK3β-KO), and GSK3α+/−/GSK3β−/− (3/4-KO). ESCs that are GSK3α−/−/GSK3β−/− were not able to differentiate into neurons (Doble et al., 2007). Since these MNs do not carry an Hb9::GFP transgene, we counted MNs as Islet (Isl)+/MAP2+ cells (Figures 6A and S6C). When deprived of neurotrophins, 25% of wild-type MNs cells survived, while 31%, 41% and 57% of MNs in GSK3α-KO, GSK3β-KO and 3/4-KO cultures survived (see Supplemental Experimental Procedures). The improved, but not completely restored, survival of cells that lack GSK-3α or/and GSK-3β again suggests that kenpaullone enhances survival by interacting with targets in addition to GSK-3. However, we cannot rule out the possibility that the incomplete rescue was due to the residual copy of GSK-3α in the 3/4-KO MNs. We also added kenpaullone to GSK-3-deficient cultures to determine whether the compound would still enhance survival. Interestingly, kenpaullone was still active, maintaining the number of MNs close to that in cultures with trophic support (Figures 6A). Thus, a variety of experimental results are consistent with the idea that GSK-3 inhibition may account for part of kenpaullone’s effect, but other activities are necessary to explain its unique set of actions.

Figure 6. see also Figure S6. Mechanistic studies of kenpaullone activity.

Figure 6

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(A) A comparison of MNs derived from wild-type, GSK3α-KO, GSK3β-KO and 3/4-KO mouse ESC lines shows that GSK-3 reduction has a small survival promoting effect when trophic factors are withdrawn. Statistics performed comparing MN survival in –TF condition of each genotype to wildtype. However, kenpaullone treatment further increased survival in all of the ESC lines, regardless of their genotype. Statistics performed comparing MN survival in +Ken treatment to −TF of the same genotype. ** p<0.01, *** p<0.0001 (Student’s t test, two-tailed). Note that MN differentiation rates across lines were comparable on the day of plating, based on Islet/MAP2 staining (data not shown).

(B-E) Kenpaullone blocks the JNK/c-Jun mediated cell death signaling cascade in MNs by inhibiting activity of an upstream activator, HGK. (B) Kenpaullone, but not CHIR 99021, blocks phosphorylation and therefore activation of c-Jun, JNK, MKK4, and Tak1 in SOD1G93A/Hb9::GFP MNs as early as 1h after treatment. * p<0.05, ** p<0.01 *** p<0.001, **** p<0.0001 (Two-way ANOVA)

(C) Examples of phospho-cJun staining (red) 24h after trophic factor withdrawal in SOD1G93A/Hb9::GFP MNs (green) treated with DMSO, 1 M CHIR 99021, or 10 M kenpaullone. The images demonstrate that kenpaullone inhibits c-Jun phosphorylation.

(D) Results from a kinase activity assay demonstrate that kenpaullone strongly inhibits HGK activity while CHIR99021 does not.

(E) A schematic of the HGK-mediated cell death pathway shows where kenpaullone acts in the signaling cascade.

Data are presented as mean ± SEM.

Additional targets of kenpaullone

We wished to identify additional molecular targets that might be acted on by kenpaullone. Since kenpaullone is reported to inhibit multiple kinases (Anastassiadis et al., 2011), we carried out preliminary studies employing a Phospho-Kinase Antibody Array to examine the site-specific phosphorylation of a variety of kinases and their protein substrates in the presence or absence of kenpaullone in MN cultures (data not shown, see supplemental experimental procedures). First, results indicated a significant reduction in relative phosphorylation of GSK-3α/β, confirming that kenpaullone inhibits activation of this kinase in our assay. Further, results indicated reduction in relative phosphorylation of both cJun N-terminal kinase (JNK) and c-Jun, respectively. Since phospho-cJun is well known to be associated with stress-induced neuronal apoptosis and can also be activated subsequent to trophic factor withdrawal (Ham et al., 1995; Watson et al., 1998), we investigated this finding more thoroughly.

For this set of experiments, we decided to compare the effects of kenpaullone to those of CHIR99021, reported to be among the most selective of GSK-3 inhibitors, yet still unable to support MN survival under our assay conditions [Figure 2A; (Bain et al., 2007)]. First, using immunostaining with phospho-specific antibodies, we observed that kenpaullone, but not CHIR99021, decreased the percent of MNs positive for phospho-cJun or phospho-JNK, even at very early time points, confirming results of the Phosho-Kinase Array (Figures 6B and 6C). We next examined MKK4, an upstream regulator of this pathway (Figure 6E) and found that its phosphorylation was also suppressed (Figure 6B).

We next sought to determine which upstream kinase in this particular pathway could be inhibited directly by kenpaullone. According to prior reports on kinase activity, potential candidates include mixed lineage kinase-3 (MLK3) and HPK1/GCK-like kinase [HGK; also known as MAP4K4; (Anastassiadis et al., 2011)]. We therefore performed in vitro phosphorylation reactions using the two recombinant kinases, with a myelin basic protein peptide as the substrate. Surprisingly, we found that kenpaullone, but not CHIR99021, differentially inhibited HGK, but not MLK3, activity (Figure 6D). Importantly, HGK is highly expressed in adult mouse motor neurons (Allen Spinal Cord Atlas; imageId=100479716). To confirm and extend these results, we performed the immunostaining studies with a site-specific antibody against phospho-Tak1, an intermediate in the cell death pathway between HGK and MKK4 (Yao et al., 1999), but uninvolved in the pathway activated by MLK3. The proportion of MNs immunopositive for phospho-Tak1 (Figure 6B) was significantly reduced by kenpaullone but not CHIR99021, incubation. Together, these results indicate that the highly beneficial effects of kenpaullone, when compared to other more specific GSK-3 inhibitors, can be attributed in part to reducing activation of the HGK-Tak1-MKK4-JNK-c-Jun cell death signaling cascade (Figure 6E).

Kenpaullone promotes survival of MNs derived from human ALS iPSCs

For our results to be relevant to treating ALS, it is important to demonstrate that they can be applied to human MNs. We first produced MNs from a human ESC line expressing an HB9::GFP transgene [HuES-3/Hb9::GFP (Di Giorgio et al., 2008)] (Figure S7) and FACS purified them to reduce the number of residual progenitor cells and other types of neurons. We found that kenpaullone treatment prevented these cells from dying after withdrawing trophic factors (Figure 7A). For comparison, we treated these cells with two compounds, olesoxime and dexpramipexole (Cudkowicz et al., 2011; Sunyach et al., 2012), that recently failed in phase III clinical trials for ALS. We found that olesoxime had some survival promoting effects, whereas dexpramipexole had no significant activity on the HuES-3 MNs (Figure 7A).

Figure 7. see also Figure S7. Effect of Kenpaullone and other ALS drugs on the survival of human MNs.

Figure 7

Open in a new tab

Fold increase in the survival of human MNs derived from (A) HuES-3/Hb9::GFP ESCs, (B) wild-type iPSCs, (C) SOD1L144F iPSCs, or (D) TDP-43M337V iPSC treated with DMSO, Kenpaullone, Olesoxime (Oles), or Dexpramipexole (Dex). Either HB9::GPF positive or Isl/Tuj1 double positive cells were counted as MNs.

Data are presented as mean ± SEM. * p<0.05, ** p<0.01, n.s., not significant, compared to –TF control (Student’s t test, two-tailed).

We then tested kenpaullone on MNs produced from iPSCs from a control individual and from two ALS patients expressing confirmed mutations in either SOD1 (Boulting et al., 2011) or in TDP-43. In all human cultures, kenpaullone produced a substantial increase in MN survival whereas dexpramipexole was virtually without effect on any of the human MN types. Olesoxime had a more positive effect, but was somewhat variable across the different MN lines (Figures 7B to 7D).

Discussion

ALS is a complicated, heterogeneous disease involving cell autonomous (MN) and non-cell autonomous (glial) elements. This, as well as the historical lack of availability of large numbers of mouse and human neural cells, has made therapeutics discovery and development difficult. For the data presented in this paper, we have taken advantage of our ability to produce large numbers of mouse MNs from ESCs and carried out survival screens using both wild-type and SOD1 mutant cells.

Our screening strategy is worthy of discussion. Recently, Höing et al. (Höing et al., 2012) described an intriguing screening assay using stem cell-derived MNs co-cultured with a microglial cell line, supporting the notion of carrying out screen using stem cell derived MNs. However, we wished to execute screens that might capture multiple forms of MN death and therefore employed a trophic factor withdrawal assay instead. We chose mouse rather than human cells to carry out our large-scale screen because cells could be rapidly produced in large numbers and carried a reporter gene, making them easily identified and quantified. Trophic factor withdrawal has been used by others to explore pathways that underlie neuronal cell death, including that of MNs (Barthélémy et al., 2004; Kieran et al., 2008). Furthermore, the cell death is rapid and extensive, making it amenable to large-scale screening. We chose to conduct our screen using both wild-type and SOD1G93A MNs since we felt this strategy would provide the best opportunity to identify hits that might act in a broadly protective way, thereby giving us a better chance of identifying molecules capable of acting therapeutically across different ALS patient populations. For this reason, we chose to follow up on compounds that were active in both MN types. Kenpaullone was one of the best of these and was able to support long-term survival under multiple conditions, impressively maintaining the integrity of the cells as assessed using a battery of electrophysiological and morphological techniques.

Previous work has shown that enhanced death of SOD1G93A/HB9::GFP, relative to HB9::GFP, MNs occurs after 2-3 weeks in culture, around the time when ALS-characteristic protein aggregates appear (Di Giorgio et al., 2007). Kenpaullone protects against this delayed form of death as well. At approximately the same time, MNs treated with kenpaullone exhibited lower levels of mutant SOD1 protein and ubiquitin, when assessed by single cell imaging. It may be that kenpaullone’s ability to increase survival of the mutant MNs is “upstream” of SOD1 aggregation or it may be that kenpaullone can prevent protein aggregation, conferring an additional boost to its effects on survival. Lowering levels of mutant SOD1 protein has been introduced as an important strategy for developing therapeutics targeted at the subpopulation of patients who have SOD1 mutations (Saito et al., 2005; Smith et al., 2006; Wang et al., 2010). Our work suggests that it may be possible to find small drug-like molecules that have this activity.

Kenpaullone is classified as a GSK-3 inhibitor. In many cell types, including neurons, GSK-3 is activated during serum/trophic factor deprivation, and inhibiting GSK-3 has been shown to reduce neuronal apoptosis (Hetman and Xia, 2000; Linseman et al., 2004). The connection between GSK-3 and ALS has been demonstrated in a number of studies, and the therapeutic potential of GSK-3 inhibitors has been discussed (Koh et al., 2007; 2011). However, our data suggest that kenpaullone acts only in part as a GSK-3 inhibitor. We tested many other GSK-3 inhibitors including those thought to be the most potent and specific, and found that most were weakly active, at best, in promoting MN survival. Furthermore, we carried out several experiments in which we reduced the expression of GSK-3 and found only relatively mild effects on survival. Kenpaullone itself was more active than any of the knockdowns and, in fact, was able to almost fully restore survival even in knockdown cells. Although these results are not conclusive since there is some residual GSK-3 activity in all of our studies, it is consistent with the possibility that kenpaullone has an additional target. To gain insight into the identity of additional kenpaullone targets, we carried out an extensive series of biochemical and immunocytochemical experiments. We demonstrated that kenpaullone inhibits HGK, a kinase expressed in MNs but never studied in the context of MN disease. Inhibition of this kinase prevents the activation of a well-known apoptosis pathway that is associated with increased nuclear levels of phospho-cJun (Yao et al., 1999). Thus, HGK potentially emerges as a new therapeutic target for treating ALS.

Finally, we conducted a small human ALS “in vitro clinical trial” in which we compared kenpaullone activity to that of two compounds that failed in the clinic. Interestingly, olesoxime had been discovered in a wildtype rat primary MN survival screen (Bordet et al., 2007), similar to one of the assays we used here. Dexpramipexole reportedly has not been tested on rodent MN cultures. Both compounds had been deemed somewhat active in the standard mouse ALS in vivo model (Bordet et al., 2007; Gribkoff and Bozik, 2008; Sunyach et al., 2012), but importantly, neither had been tested on human MNs of any variety. Even our small trial shows that it is possible to detect important functional differences among these compounds when they are tested on human MNs. For example, dexpramimpexole was totally ineffective on any of the human MN lines, and our results might have predicted its lack of efficacy in the clinic, whereas olesoxime had less consistent activity and somewhat smaller effects. Kenpaullone, therefore, emerges as a preclinical compound of interest, but requires additional chemical modification to improve its potency and CNS penetration. Alternatively, it may be possible to find an inhibitor that acts selectively on HGK kinase and that will have sufficient therapeutic activity. We predict that more comprehensive versions of our in vitro clinical trial in which compounds are tested on many lines of human diseased cells, such as MNs, will be extremely useful in choosing the best compounds to take into clinical studies and in selecting patients most likely to respond to particular treatments (Rubin, 2008).

Experimental Procedures

Chemicals

Kenpaullone, LY294002 and PI-103 were purchased from Tocris. Alsterpaullone, GSK-3 inhibitor VIII and GSK-3 inhibitor IX were purchased from Millipore. CHIR 99021 was purchased from Stemgent. CHIR 98014 was from Axon Medchem. Ara-C was from Sigma-Aldrich.

Mouse ESC Culture and MN Differentiation

Mouse ESCs were differentiated into MNs and cultured as described (Makhortova et al., 2011) and plated in polyornithine-coated 96-well plates or poly-D-lysine-coated 384-well plates. For some experiments, MNs were purified by FACS, using a MoFlo XDP (Beckman Coulter) flow cytometer to isolate GFP+ cells. FACS purified MNs were plated on growth factor reduced Matrigel (BD Biosciences).

Mouse MN Survival Assay

For the small molecule screen or for standard survival assays, freshly dissociated EBs were plated at a density of 8,000 GFP+ cells (384-well) or 30,000 GFP+ cells (96-well) per well. Four days later, trophic factors were removed, and individual compounds were added to the wells. For the primary screen each compound was tested at three concentrations (0.1μM, 1μM and 10μM) in duplicate. After an additional 72 hours (day 7), cells were fixed in 4% paraformaldehyde, stained with Hoechst 33342 (Life Technologies) and scanned using an automated confocal microscope (PerkinElmer Opera) at 10× magnification. The number of MNs surviving was analyzed by counting the remaining GFP+ cells in the whole well. Total cell numbers were obtained by counting Hoechst-positive nuclei. A size threshold was set based on control cultures to eliminate apoptotic MNs. Unless stated otherwise, survival is measured as fold-increase compared to cultures maintained without trophic factors.

For long-term MN survival assays, MN cultures were fed with fresh medium and compounds every two days until fixation and quantification as above. For gene-specific knockdown with shRNAs, plated MNs were infected with lentivirus carrying shRNAs on day 2, trophic factors were withdrawn on day 4, and cells were fixed on day 7.

Human MN Differentiation and Survival Assay

Human MNs were derived from human ESCs and iPSCs as previously described (Ding et al., 2013). After 30 days of differentiation, the cultures were dissociated with papain solution (Worthington) and plated as single cells on 384-well plates (Greiner) pre-seeded with mouse astrocytes. The medium used was Neurobasal containing 2% B27 and 1% N2 (Life Technologies) supplemented with 20 ng/mL of BDNF, GDNF and CNTF (R&D Systems). For human MN survival assays, trophic factors were removed, compounds were added on day 6, and cells were fixed after a further 14-day incubation. Ara-C was present throughout the entire experiment to eliminate proliferating progenitors. MN survival was determined by counting Hb9::GFP+ cells (HuES-3/Hb9::GFP) and Isl+/MAP2+ or Isl+/Tuj1+ cells (human iPSCs).

Immunocytochemistry

Immunostaining was carried out using standard protocols (Makhortova et al., 2011). The following primary antibodies were used: mouse anti-Islet (DSHB), mouse or rabbit anti-Tuj1 (Covance), rabbit anti-MAP2 (EMD Millipore), rabbit anti-synapsin (EMD Millipore), mouse anti-PSD95 (BD Biosciences), mouse anti-hSOD1 (Sigma-Aldrich), goat anti-mSOD1 (R&D SYSTEMS), rabbit anti-ubiquitin (DAKO), rabbit anti-Olig2 (EMD Millipore), anti-BrdU-Alexa 555 (BD Biosciences) rabbit anti-phospho-cJun (Cell Signaling), mouse anti-phospho-JNK (Cell Signaling), rabbit anti-phospho-MKK4 (Cell Signaling), and rabbit anti-phospho-Tak1 (Cell Signaling). Secondary antibodies conjugated with Alexa 488, Alexa 546 or Alexa 647 were purchased from Life Technologies. Hoechst 33342 was used for nuclear staining.

Electrophysiology

Whole-cell voltage-clamp and current-clamp recordings were performed as described (Son et al., 2011).

Statistical Analysis

Statistical significance was determined by Student’s t-test for two groups or by ANOVA for three or more groups. A CI of 95% was used for all comparisons. Results are reported mean ± SD or mean ± SEM.

Supplementary Material

01

Highlights.

  • A new type of stem cell based motor neuron screen was performed.

  • Kenpaullone increases the survival of wildtype and ALS motor neurons.

  • Kenpaullone’s activities result from dual inhibition of GKS-3 and HGK kinases.

  • Potential value of preclinical testing using human ALS motor neurons is shown.

Acknowledgements

We are grateful to K. Eggan for providing mouse ESCs and human ESCs and iPSCs, G. Daley for providing the TDP-43 human iPSCs, and B. Doble and J. Woodgett for providing mouse GSK-knockout ESC lines. We would also like to thank B. Tilton and P. Rogers for assistance with FACS, K. Kotkow A.D. Sinor-Anderson, N. Makhortova, M. Hayhurst, K. Krumholz, G. Boulting, J. Ichida, E. Kiskinis, and A. Blumenstein for providing reagents and helpful discussions, and J. LaLonde for editorial assistance. This work was supported by the ALS Association, NINDS (P01NS066888), the New York Stem Cell Foundation, and the Harvard Stem Cell Institute.

LLR is a founder of iPierian Inc. and a member of its SAB.

Footnotes

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