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Aging is a risk factor for the development of adult-onset neuro-degenerative diseases. While some of the molecular pathways regulating longevity and stress resistance in lower organisms are defined (i.e., those activating the transcriptional regulators DAF-16 and HSF-1 in C. elegans), their relevance to mammals and disease susceptibility are unknown. We studied the signaling controlled by the mammalian homolog of DAF-16, FOXO3a, in model systems of motor neuron disease. Neuron death elicited in vitro by excitotoxic insult or the expression of mutant SOD1, mutant p150glued or polyQ expanded androgen receptor was abrogated by expression of nuclear-targeted FOXO3a. We identify a compound (Psammaplysene A, PA) that increases nuclear localization of FOXO3a in vitro and in vivo and show that PA also protects against these insults in vitro. Administration of PA to invertebrate model systems of neurodegeneration similarly blocked neuron death in a DAF-16/FOXO3a-dependent manner. These results indicate that activation of the DAF-16/FOXO3a pathway, genetically or pharmacologically, confers protection against the known causes of motor neuron diseases.
Although motor neuron diseases due to single gene mutations are unusual (~10% of cases), the affected genes have been successfully used to model these diseases in experimental systems (Cleveland and Rothstein, 2001). Rare, genetic forms of motor neuron disease that arise from mutations in superoxide dismutase (SOD) or p150glued have a disease phenotype that strongly resembles sporadic ALS with lower motor neuron predominance (Pasinelli and Brown, 2006). Another predominantly lower motor neuron disease, called spinobulbar muscular atrophy (SBMA or Kennedy's Disease) is due to a polyglutamine expansion in the androgen receptor (La Spada et al., 1991). Despite identification of the “disease protein”, a unifying pathogenic mechanism has not been defined.
In most cases of sporadic ALS, motor neuron death is triggered by the interaction of a genetic pre-disposition and environmental factors (Bruijn et al., 2004). Genome-wide association studies have failed to reveal consistent susceptibility loci (Dunckley et al., 2007; Kasperaviciute et al., 2007). Correlative evidence suggests that aging is a risk factor for the development of ALS as well as other adult-onset neurodegenerative disorders (Lin and Beal, 2006). Studies from a variety of experimental systems have provided insight into the genetic factors controlling aging, in particular, the insulin/insulin-like growth factor signaling pathway (Hekimi and Guarente, 2003; Longo and Finch, 2003). In Caenorhabditis elegans, hypomorphic alleles of the daf-2 gene (mammalian homolog, insulin/insulin-like growth factor receptor) and the downstream signaling molecule age-1 (mammalian homolog, phosphotidylinositol-3′-kinase, PI3′K) promote longevity and lifespan extension requires the activity the DAF-16 transcription factor (mammalian homolog, FOXO3a)(Tatar et al., 2003).
DAF-16/FOXO3a shuttles between the cytoplasm (where it is inactive) and the nucleus in a process that is controlled by its phosphorylation state (Brunet et al., 1999). Phosphorylation of DAF-16/FOXO3a by the PI3′K substrate kinases Akt and SGK (Brunet et al., 2001) leads to the 14-3-3 protein-dependent export of nuclear DAF-16/FOXO3a and reentry into the nucleus requires dephosphorylation and release of 14-3-3 (Brunet et al., 2001; Greer and Brunet, 2005). Within the nucleus, DAF-16/FOXO3a leads to the expression of a number of genes that can help cope with stress and play an essential role in its longevity promoting activity (Birkenkamp and Coffer, 2003). Reducing the activity of the insulin/insulin-like growth factor signaling (IIS) pathway leads to increased DAF-16/FOXO3a-dependent transcription and enhanced stress resistance. In a nematode model of Aβ1-42 toxicity, reduction of IIS is neuroprotective (Cohen et al., 2006).
Some of the stresses that contribute to motor neuron death in ALS include excitotoxicity, reactive oxygen species, accumulation of insoluble aggregates of neurofilaments, and defects in axonal transport (Cleveland and Rothstein, 2001; Rao and Weiss, 2004; Boillee et al., 2006; Lobsiger and Cleveland, 2007). Since DAF-16/FOXO3a-dependent gene transcription, in other contexts, combats cellular stresses, we inquired whether manipulating FOXO3a signaling protected neurons from insults relevant to motor neuron diseases. We show FOXO3a activation is neuroprotective across phyla and identify a toxicity-sparing pharmacological approach for enhancement of DAF-16/FOXO3a signaling activation.
In fibroblasts, expression of FOXO3a with alanine substitutions at three phosphorylation sites (Thr-32, Ser-253 and Ser-315) leads to nuclear retention of the transcriptionally active protein (Brunet et al., 2001). Cultures of rat spinal cord neurons were infected with recombinant HSV engineered to express the triple mutant (TM) or wild type (WT) FOXO3a. Immuno-staining for the transgene demonstrated that the WT-FOXO3a is restricted to the neuronal cytoplasm and the TM-FOXO3a is largely nuclear (Figure 1A). The transgene products were detectable in all neurons for >5 days without any apparent toxicity. To determine if these transgenes influenced the susceptibility of motor neurons to excitotoxic insult, 14 days in vitro (DIV) mixed spinal cord cultures were infected with HSV- WT-FOXO3a, HSV- TM-FOXO3a, HSV-LacZ or no virus, the following day exposed to an excitotoxic challenge (100 μM kainic acid (KA) or vehicle for 1h) and the number of surviving motor neurons determined 24 hours later (Fryer et al., 1999; Fryer et al., 2000). While approximately 45% of motor neurons were killed by KA in the HSV-LacZ and HSV-WT-FOXO3a infected cultures, no KA-induced motor neuron death occurred in the HSV-TM-FOXO3a infected cultures (Figure 1B). ANOVA using transgene expression as the between-group factor and survival as the within-group factor demonstrated a significant difference between groups (F(5,12) = 15.68; p< 0.001, ANOVA). The post-hoc comparisons between groups using Scheffé's F test with significance set at p < 0.05 showed that significant motor neuron death only occurred in the cultures expressing the WT-FOXO3a or LacZ. Thus expression of the TM-FOXO3a protects cultured motor neurons from excitotoxic insult.
Next we explored the capacity of TM-FOXO3a to protect motor neurons in vitro from a variety of proteotoxic insults relevant to motor neuron diseases. In spinobulbar muscular atrophy a polyglutamine expansion in the androgen receptor (AR) leads to testosterone-dependent motor neuron death (Chevalier-Larsen et al., 2004). We made cultures from spinal cord of mutant AR-expressing mice and found that a 7 day treatment with dihydrotestosterone (DHT) led to the loss of approximately 25% of motor neurons compared with the vehicle-treated cultures (p < 0.05) (Figure 1C). We observed an equivalent rate of DHT-dependent motor neuron death in cultures treated with HSV-LacZ or HSV-WT-FOXO3a (p < 0.05). Infection of cultures with HSV-TM-FOXO3a, however, completely blocked DHT-dependent death of motor neurons, with all motor neurons surviving in the presence of DHT (Figure 1C).
Proteotoxic motor neuron death can also be precipitated by expression of mutant forms of human superoxide dismutase or p150glued (Mojsilovic-Petrovic et al., 2006a). We asked if treating cultures expressing these mutant proteins with TM-FOXO3a affected motor neuron death. We began these studies by determining concentrations of viruses that led to co-expression of both transgenes in neurons but did not lead to toxicity owing to the viral burden. We established that infection of spinal cord neurons with ~8 × 104 pfu of HSV-mutant SOD/ml culture media reliably induced 50% motor neuron loss 7 days post infection. Addition of ~8 × 104 more pfu of HSV-LacZ to these wells did not exacerbate motor neuron death. Immunocytological localization studies revealed that 97 ± 2 % of motor neurons expressing mutant SOD also expressed β-galactosidase (Figure 1D). In all subsequent studies we confirmed a >95% co-expression of the toxicity-inducing mutant protein (SOD or p150glued) and LacZ or TM-FOXO3a. We also established that co-expression of LacZ with either WT SOD or WT p150glued had no adverse effect on motor neuron survival. We previously have shown that this concentration of HSV-mutant SOD infects all motor neurons at this plating density (Hu and Kalb, 2003; Mojsilovic-Petrovic et al., 2006a).
We compared next the outcome of cultures infected with HSV-mutant SOD + HSV-LacZ or HSV-TM-FOXO3a as well as cultures infected with HSV- mutant p150glued + HSV-LacZ or HSV-TM-FOXO3a (Figure 1E). The ANOVA indicates that statistically significant differences between groups existed (F(5,12) = 70.14, p < 0.0001). Infection of cultures with HSV-mutant SOD + HSV-LacZ led to significantly lower motor neuron numbers when compared with cultures infected with HSV-mutant SOD + HSV-TM-FOXO3a (47.0 ± 4.5 versus 90.6 ± 1.5, p < 0.05 in post hoc analysis). Similarly motor neuron numbers from cultures infected with HSV- mutant p150glued + HSV-LacZ were significantly lower than motor neuron numbers from cultures infected with mutant p150glued + HSV-TM-FOXO3a (42.3 ± 4.0 versus 91.3 ± 3.7, p < 0.05 in post hoc analysis) (Figure 1E). Thus the toxicity of three different mutant proteins that cause motor neuron disease can be blocked by expression of a version of FOXO3a that constitutively resides in the nucleus.
A chemical-genetic screen recently reported the identification of a series of compounds that can inhibit FOXO1a nuclear export. Compounds fell into two classes: 1) inhibitors of general nuclear export machinery and 2) inhibitors specific to the PI3′K/Akt/FOXO1a pathway (Kau et al., 2003). We inquired whether compounds in the second class would also block the nuclear export of FOXO3a since they would be predicted (based on the results above) to display neuro-protective activity. We focused on Psammaplysene A (PA), the most potent of the class 2 inhibitors, which was isolated from a marine sponge (Figure 2A) (Schroeder et al., 2005).
We began by looking at the effect of a synthetic sample of PA on the distribution of FOXO3a endogenously expressed by neurons (Georgiades and Clardy, 2005). In vehicle treated cultures, FOXO3a was cytoplasmic and clearly excluded from the nucleus. In contrast, 24 hour treatment of cultures with 10 nM PA led to strong nuclear localization of FOXO3a (Figure 2B). We next biochemically isolated nuclei from spinal cord cultures treated with PA or vehicle (Figure 2C). Based on the distribution of the nuclear envelope protein lamin, it is clear our subcellular fractionation procedure greatly enriched nuclei. There was an ~ 2.5 fold increase in the nuclear FOXO3a/nuclear lamin ratio in the PA treated cultures in comparison to vehicle treated cultures. Total FOXO3a and lamin levels were unaffected by drug treatment. These observations indicate that PA promotes the sequestration of FOXO3a into nuclei.
To determine if PA had neuroprotective activity, spinal cord cultures were treated with the drug (10 nM) for two days and then subjected to an excitotoxic challenge (Figure 2D). The percent of KA-induced cell death was 55 ± 4 % in vehicle treated cultures and 3 ± 1 % in PA treated cultures (p < 0.01, Student's t-test) indicating that PA protected motor neurons from excitotoxic challenge. Next we looked at mutant AR proteotoxicity (Figure 2D). Significant differences between groups (F(2,6) = 18.84, by ANOVA) were found in the three-way comparison of 1) No DHT, 2) DHT + vehicle, and 3) DHT + PA). The post hoc analysis demonstrated a DHT-dependent ~30% loss of motor neurons in vehicle treated cultures (p < 0.01) and neuroprotection in the PA treated cultures (P > 0.05 in the comparison of no DHT versus DHT + PA).
We followed up these observations by asking if PA blocked the proteotoxicity of SOD or p150glued. Spinal cord cultures were infected with HSV engineered to express the WT or mutant forms of SOD or the WT or mutant forms of p150glued and received PA (or vehicle) every other day for 4 days. The drug treatment had no effect on transgene expression (not shown). After 4 days, the cultures were fixed and motor neuron number was determined. ANOVA revealed statistically significant differences between groups in LacZ versus WT SOD versus mutant SOD (± PA) comparisons (F(5,12) = 18.41, p < 0.001) as well as LacZ versus WT p150glued versus mutant 150glued (± PA) comparisons (F(5,12) = 19.26, p < 0.001) (Figure 2E). The post hoc analysis revealed that statistically significant protection against the toxicity of mutant SOD or p150glued was conferred by PA treatment on motor neuron survival. PA had no adverse effect on survival on motor neurons expressing LacZ or wild type versions of SOD or p150glued. Thus PA is non-toxic on its own but can protect against four different insults in vitro that are directly relevant to motor neuron diseases.
Although the direct molecular target of PA is unknown, we examined the effect of PA on some candidate biochemical and cell biological processes that have previously been implicated in neuron death. Neurotrophins (such as brain-derived neurotrophic factor, BDNF) can promote neuronal survival by activating its receptor TrkB both during development and after insult ((Koliatsos et al., 1993; Ernfors et al., 1995; Klocker et al., 1998), but see (Koh et al., 1995; Mojsilovic-Petrovic et al., 2006a)) and so we wondered if PA had demonstrable effects this signaling pathway. Spinal cord cultures grown for 14 DIV were infected with HSV-mutant SOD and then PA or vehicle was added to the cultures. Under these conditions, in the absence of BDNF, the level of active, phosphoTrk receptor is very low (Figure 3A). Similarly, downstream signaling involving AKT and MAPK are only modestly active. In response to BDNF addition to the media, there is a rapid and robust activation of the Trk receptor (as monitored by assaying for the phosphorylated form of the receptor) as well as phosphoAKT and phosphoMAPK. The temporal pattern of receptor activation and downstream signaling in our cultures conforms to previous observations (Fryer et al., 2000; Hu and Kalb, 2003) (Figure 3A). Identical results were obtained in cultures uninfected with viruses (not shown). Thus we find no evidence that pre-treatment of cultures with PA has any effect on the magnitude or duration of BDNF-TrkB signaling.
Insoluble aggregates of mutant SOD are detectable within cells from transgenic mice engineered to express mutant SOD (Bruijn et al., 1997; Watanabe et al., 2001). We wondered if WT or mutant SOD aggregated in neurons in vitro and if treating cultures with PA influenced the cellular distribution of transgene human SOD. Immunocytological location of human SOD in cultures infected with HSV-WT-SOD revealed that the protein is homogeneously distributed throughout the cytoplasm and extends centrifugally for >100 microns into axons and dendrites (Figure 3B). In contrast, in cultures infected with HSV- G87R human SOD immunoreactivity is concentrated into puncta (the cytological signature of insoluble aggregated proteins) in the soma cytoplasm and neurites. Double labeling studies reveal that mutant SOD puncta are present in motor neurons (identified by SMI32 immunoreactivity) as well as non-motor neurons in our cultures (Figure 3C). Treatment of cultures with PA had no effect on the subcellular distribution of human SOD in cultures infected with either of the recombinant HSVs. Although the pathophysiological significance of aggregated protein is controversial, these results indicate that the neuroprotective action of PA is dissociable from the accumulation of aggregated mutant SOD. A similar observation has been made in C. elegans wherein DAF-16 protects against Aβ1-42 toxicity but does not influence the accumulation of protein aggregates (Cohen et al., 2006).
Since the neuroprotection conferred by PA does not seem to be linked to alterations in trophic factor signaling or the generation of macroscopic mutant SOD aggregates, perhaps PA action is linked to its capacity to promote nuclear localization of FOXO3a. This would be consistent with the observations that PA causes nuclear partitioning of FOXO3a in neurons and constitutive localization of FOXO3a in the nucleus is broadly neuroprotective. Evidence in favor of this view comes from biochemical studies of spinal cord neurons in vitro expressing LacZ, WT-SOD or G87R-SOD. In the absence of PA, the abundance of phosphorylated FOXO3a (or its ratio to the non-phosphorylated species) is greatly enhanced in G87R-SOD, in comparison with LacZ or WT-SOD, expressing cultures (Figure 3C). One transcriptional target of FOXO3a is MnSOD and the abundance of this protein is markedly depressed in G87R-SOD, in comparison with LacZ- or WT-SOD-expressing cultures. These observations are consistent with the view that FOXO3a phosphorylated at Thr-32, Ser-253 and Ser-315 is cytoplasmic and thus unable to participate in promoting MnSOD expression (Brunet et al., 1999). None of the recombinant viruses led to measurable changes in total FOXO3a or actin. In contrast with these observations, PA treatment rescued all of the biochemical alterations (Figure 3C). There were no differences in the abundance of phosphoFOXO3a (or its ratio to the non-phosphorylated species) or in the abundance of MnSOD between any of the three experimental groups. These findings suggest that mutant SOD proteotoxic stress is associated with a change in the state of phosphorylation of FOXO3a in a manner that can lead to nuclear exclusion and diminution in the abundance of one of its transcriptional targets. Precisely how PA effects changes in the state of FOXO3a phosphorylation and whether the effects of PA on MnSOD occur at the transcriptional level remain to be explored. These observations nevertheless suggest that proteotoxic stresses induce changes in the FOXO3a signaling pathway.
Given these in vitro observations, we wondered if a similar phenomenon occurs in mutant SOD mice. We examined lysates from spinal cords of mice expressing the G93A mutant form of human SOD or wild type controls for the expression of phosphoFOXO3a and target genes. The mice were 87 days old, a time when they are asymptomatic in terms of weakness, but do manifest other subtle abnormalities (Mourelatos et al., 1996; Frey et al., 2000; Pun et al., 2006). A consistent increase in phosphoFOXO3a (and the ratio of phosphoFOXO3a to the non-phosphorylated species) was seen in the G93A mice in comparison with the wild type animals (n=4 in each experimental group) (figure 3D). This was associated with a reduction in MnSOD in the G93A mice in comparison with the wild type animals. No differences were noted in the abundance of actin in the mutant versus wild type animals. Thus a reduction in the abundance of a FOXO3a transcriptional target was detected in the spinal cord of pre-symptomatic mutant SOD mice, raising the possibility that abnormal FOXO3a signaling might contribute to disease pathogenesis.
To further explore this issue we employed bioinformatics tools to query existing microarray profiles for a potential connection between motor neuron disease and FOXO3a-dependent transcription. We identified five microarray datasets located in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) server that are relevant to motor neuron disease. These datasets come from studies of postmortem tissue from ALS patients and mutant SOD mouse or rat tissues at various stages of disease. To our knowledge the only description of the FOXO3a transcriptome comes from a study of PTEN null 786-O renal carcinoma cells (Ramaswamy et al., 2002). This study identified 198 transcripts whose level of expression is changed ≥2-fold when a DNA binding-competent, constitutively nuclear (triple mutant) FOXO3a is expressed in these cells. When we asked how many genes in the motor neuron disease data set were also components of the FOXO3a transcriptome, we identified 22 transcripts in both datasets (Supplemental Table). Even given the disparate experimental platforms employed in these studies, the presence of any overlapping dataset is intriguing. A better-controlled prospective study is required to obtain a more full understanding of the potential importance of FOXO3a in motor neuron disease pathogenesis.
In light of the neuroprotective effect of PA in vitro, we examined the effects of the drug in two in vivo model systems of neuronal degeneration. Expression of polyglutamine-expanded AR in the Drosophila eye leads to DHT-dependent degeneration (Takeyama et al., 2002; Pandey et al., 2007). We found that flies reared on food supplemented with 0.5 mM PA had a reduced degenerative phenotype when compared with vehicle treated flies (Figure 4 A- B). These in vivo results complement the observations made in spinal cord cultures from mice expressing polyglutamine-expanded AR wherein we found expression of TM-FOXO or treatment with PA blunts DHT-dependent mutant AR toxicity.
We next asked if the neuroprotective action of PA depended on FOXO. To this end, we examined the efficacy of PA in flies that both expressed the polyglutamine expanded AR and were haploinsufficient for Drosophila Foxo (dFoxo)(Junger et al., 2003) (Figure 4 C-D). On a dFOXO deficient background (Foxo21-allele), PA lost its ability to protect against DHT-dependent degeneration. To be sure this observation was not due to genetic background issues, we studied a second dFOXO loss-of-function allele (Foxo25). As above, on this dFOXO deficient background (Foxo25-allele), PA lost its ability to protect against DHT-dependent degeneration (Figure 4 E-F). Using a quantitative rating score, we found that PA led to a statistically significant mitigation of polyglutamine-expanded AR degeneration but this was lost in the dFOXO haploinsufficient flies (Figure 4 G). These observations indicate that PA confers protection against mutant AR proteotoxicity in a FOXO-dependent manner.
We developed a C. elegans model system of neurodegeneration by combining a null mutation in the glutamate transporter glt-3 (•glt-3) with a transgenic strain (nuIs5 - (Berger et al., 1998)) in which the glr-1 promoter drives expression of an activated form of Gs (abbreviated Gs*) and GFP in glutamatergic neurons (Mano and Driscoll, 2005; Mano et al., 2007). The •glt-3;nuIs5 double mutants demonstrate necrotic neuron death at all stages of postembryonic development, with the strongest effect seen in developmental stage L3. PA had a dose-dependent neuroprotective effect at the L3 stage with complete rescue from death using 10 nM PA (Figure 5A). This concentration of PA had no adverse effect on WT nematodes. We asked if the effect of PA is mediated by changing the timing of neurodegeneration or by reducing it throughout development (Figure 5B). To examine this we studied the effect of PA on the •glt-3;nuIs5 double mutants as a function of larval stage and we found neuronal death was reduced in all developmental stages, with the strongest effect observed in the developmental stages most prone to excitotoxicity. Neuron death was reduced in a statistically significant manner in larval stages L2 (3.4 ± 0.2 versus 2.5 ± 0.2 dying neurons/animals, n = 44 versus n = 49, vehicle versus PA, p = 0.006) and L3 (4.2 ± 0.2 versus 2.2 ± 0.1 dying neurons/animals, n = 63 versus n = 65, vehicle versus PA, p < 0.001) (Figure 5B).
In C. elegans, stress resistance and longevity is promoted by a reduction in the activity of insulin growth factor receptor (IGFR) signaling pathway (i.e., hypomorphic alleles of daf-2, the IGFR and age-1, nematode PI3′K) and this requires daf-16, the nematode homolog of FOXO3a. This led us to wonder if reducing activity in the IGFR signaling pathway would alleviate nematode excitotoxicity. To that end, we crossed the •glt-3;nuIs5 double mutants with an age-1 mutants that carry the hx546 allele. This allele has a specific anti-aging effect but does not affect development (Friedman and Johnson, 1988). We generated triple mutant nematodes (age-1; • glt-3;nuIs5) and found a robust neuroprotective effect of age-1 at larval stages L1, L2 and L3 (all p values • 0.001) (Figure 5C).
Finally, while we showed that PA leads to accumulation of FOXO3a in mammalian neuronal nuclei, we wished to determine if the same was true in C. elegans. To this end, we studied nematodes in which a DAF-16::GFP fusion protein was expressed in body wall muscles. Addition of PA, but not vehicle, to the growth substrate led to nuclear localization of the fusion protein and quantification of the nucleus/cytoplasm ratio of DAF-16::GFP revealed a statistically significant effect of PA (1.46 ± 0.05 versus 3.44 ± 0.55, n = 36 versus n=27, vehicle versus PA, p < 0.0001) (Figure 5D). This result indicates that PA has an evolutionarily conserved capacity to promote nuclear localization of the DAF16/FOXO3a transcription factor and this is associated with resistance to necrotic neuron death. Another method for promoting nuclear localization of DAF-16 (mutation of age-1) has a neuroprotective effect and further re-enforces the notion that manipulation of the IGFR signaling pathway could have promise as a neuroprotective strategy.
Biochemical pathways that regulate longevity in yeast, C. elegans, Drosophila melanogaster and mice also play a fundamental role in resistance to stresses such as UV radiation, oxidative conditions, heat shock and misfolded and aggregation-prone proteins (Finkel and Holbrook, 2000). Two transcription factors, Heat-shock factor 1 and DAF-16/FOXO3a, are essential mediators of this longevity/stress resistance program in nematodes (Hsu et al., 2003; Morley and Morimoto, 2004). Here we show that genetic and pharmacological maneuvers that enhance nuclear localization of FOXO3a protect mammalian motor neurons in vitro from 4 insults directly relevant to motor neuron diseases and abrogate neurodegeneration in two in vivo invertebrate models systems. The broad neuroprotective action of PA suggests it acts on a phylogenetically conserved, core stress resistance pathway.
Several lines of evidence suggest that the neuroprotective actions of PA are mediated, at least in part, by FOXO3a/DAF16. First, in both rat neuron cultures and C. elegans, application of PA promotes nuclear accumulation of FOXO3a or DAF16. Second, constitutive nuclear localization of FOXO3a mimics the neuroprotective action of PA. Third, expression of mutant SOD (both in vitro and in vivo) provokes an increased phosphoFOXO3a/FOXO3a level (which leads to cytoplasmic sequestration of FOXO3a) and this results in decreased abundance of MnSOD (a known transcriptional target of FOXO3A). Fourth, PA-treated cultures expressing G87R mutant SOD exhibit restored phosphoFOXO3a/FOXO3a ratio and lead to an increased abundance of MnSOD. Fifth, PA-mediated protection against mutant AR evoked degeneration is dFOXO-dependent. Even without knowledge of the direct molecular target of PA, the extant evidence strongly favors the view that the neuroprotective action of PA is mediated by a FOXO3a transcription-dependent manner.
Upon nuclear localization, members of the FOXO family of transcription factors control the expression messages that impact glucose metabolism, tumor suppression (through effects on cell cycle progression and apoptotic responses), stress resistance and longevity (Ramaswamy et al., 2002; Greer and Brunet, 2005). The specific effects of FOXOs are both cell-type and context-dependent and specific posttranslational modifications (i.e. phosphorylation, ubiquitination, acetylation) play a key role in controlling the transcriptional readout (Brunet et al., 1999; Brunet et al., 2004; Hu et al., 2004). For example, work from the Brunet lab has demonstrated that AMPK phosphorylates FOXO3a at 6 sites and the state of phosphorylation at these sites does not affect nuclear localization (Greer et al., 2007a). Instead these sites are vital for expression of genes involved in oxidative stress management and energy metabolism. Our finding that induction of MnSOD by PA in cultures expressing mutant SOD parallels the observation of Greer et al. that AMPK induction of sod-3 expression in nematodes is FOXO/DAF-16-dependent (Greer et al., 2007b). Our preliminary studies suggest that expression of mutant SOD ± PA does not influence the state of phosphorylation of FOXO3a at S413 or S588 (unpublished observations). Further work is required to determine if FOXO3a undergoes posttranslational modifications in cells expressing mutant SOD, if PA influences such a change, and whether this has functional consequences.
It is noteworthy that expression of mutant SOD in neurons in vitro or in vivo prior to neuron loss is associated with an increase in FOXO3a phosphorylation at T32 (one of the three key sites controlling nuclear/cytoplasmic partitioning) and a reduction in the abundance of MnSOD. These observations may indicate that as a consequence of expressing mutant SOD, neurons upregulate the activity of FOXO3a kinases (and/or decrease the activity of FOXO3a phosphatases) and this results in a reduction in the expression of FOXO3a regulated transcripts that combat proteotoxicity. In fact, there is a 2.5 fold increase in PI3′K activity and protein in spinal cords of ALS patients (the activator of the FOXO3a kinases AKT and SGK (Wagey et al., 1998)) as well as a 3.6 fold increase in phosphoAKT itself in mutant SOD mice (Hu et al., 2003). These observations may indicate that a putatively beneficial response to stress, such as increased expression and activity of an anti-apoptotic pathway, may have an unintended adverse additional action on FOXO3a transcription.
Given the potent neuroprotective activity of PA in vitro we sought to examine its effects in vivo. The two model systems we chose have features that are reminiscent of human motor neuron diseases. Neurons in the •glt-3:nuIs5 nematode die an excitotoxic cell death and there is abundant evidence that implicate excitotoxic mechanisms in ALS (Rao and Weiss, 2004). Spinobulbar muscular atrophy is caused by expression of the proteotoxic polyQ expanded AR and transgenic expression of this mutant in the fly eye similarly causes degeneration (Pandey et al., 2007). The neuroprotective activity of PA in these model systems complements the in vitro studies with mammalian motor neurons. A simple explanation for these observations is that PA operates on a biochemical pathway common in all these experimental systems that can overcome a diversity of noxious insults.
Several considerations dissuaded us from attempting to study the action of PA in the mutant SOD mouse model system of motor neuron disease. Significant controversy surrounds the mutant SOD mouse and many researchers view these mice as a “pathway model” as opposed to a “disease model” (Benatar, 2007; Schnabel, 2008; Scott et al., 2008). In practice this means that it may not be possible to derive reliable survival data from a drug study. In fact at least one drug (minocycline) that promoted longevity in the mutant SOD mouse (Kriz et al., 2002) appears to accelerate the clinical course of human ALS (Gordon et al., 2007). Given these considerations, we focused our work in a more tractable direction.
The onset of many neurodegenerative diseases in adulthood raises the possibility that the causes of aging are specific contributors to disease pathogenesis. To the extent that this is true, applying our understanding of the molecular biology of longevity may lead to new types of therapy for neurodegenerative diseases. The present work on FOXO3a is an example of how manipulation of a phylogenetically conserved, longevity-promoting signaling pathway can effectively block neurodegeneration. This is particularly interesting in light of a recent population-based study of human longevity showing a strong association of FOXO3a and healthy aging (Willcox et al., 2008). Agents that trigger neurodegeneration may act in part by subvert the normally healthful actions of an aging pathway.
Trophic factors (ciliary neuronotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT 4), cardiotrophin 1 (CT 1) and glial-derived neurotrophic factor (GDNF)) were obtained from Alomone Labs (Jerusalem, Israel). Psammaplysene A was synthesized as described (Georgiades and Clardy, 2005). All other reagents were obtained from Sigma (St. Louis, MO) and were of the highest grade available.
Embryonic Sprague-dawley rat spinal cord neurons were grown on confluent monolayers of cortical astrocytes, as previously described (Hu and Kalb, 2003). The substratum was acid washed glass coverslips when imaging was performed and Primaria tissue culture plasticware (Falcon, Becton-Dickinson) when biochemistry was performed. Culture media consisted of astrocyte-conditioned media supplemented with 10 ng/ml CNTF, BDNF, NT 4, CT 1 and GDNF and 50% of media was replaced with fresh media every 3 days.
Male SBMA mice transgenic for the prion protein promoter-driven androgen receptor cDNA containing an expanded (112) CAG repeat tract were mated with non-transgenic C57Bl/6 female mice (Chevalier-Larsen et al., 2004). Dissociated, mixed spinal cord cultures obtained from 13.5-day embryos were grown for 3 weeks in conditioned medium (conditioned on normal mouse astrocytes) containing charcoal-stripped serum to remove hormones. At 3 weeks, motor neurons were well differentiated and distinguishable from other neurons, including sensory neurons, by size and morphology. At this time, cultures were treated with indicated conditions for 7 days; cells were then fixed with 4% paraformaldehyde and immunostained using antibodies to neurofilament heavy chain (NF-H) (SMI32; Sternberger Monoclonal Inc.). Motor neurons were visualized using a Leica DMR fluorescence microscope, photographed, and analyzed using IP Labs software. Statistical analyses of results were carried out using Student's t-test (viral infections) or ANOVA (compound treatments; SigmaStat).
cDNAs were cloned into the PrpUC amplicon plasmid to generate recombinant HSV as previously described (Neve et al., 1997). The titer of virus used in these studies was routinely 3-5 × 107 plaque-forming units/ml. The sources of constructs were: Michael Greenberg, Harvard University (HA-tagged wild type and triple mutant human FOXO3a), David Borchelt, University of Florida (WT and mutant SOD), Erika Holzbaur, University of Pennsylvania (WT and mutant p150glued).
After 14 days in vitro (DIV), culture media was removed (and saved) and cells were exposed to 100 μM kainic acid (KA) for 1 hour. Subsequently they were washed three times in Locke's buffer not containing KA, the original media was replaced and incubated for another 24 hours at 37 °C. in 5% CO2 before fixation in 4% paraformaldehyde. Motor neurons were identified in mixed culture by immunostaining for nonphosphorylated neurofilaments and counting only labeled cells with cell body diameter of 25 μm or greater. We have previously validated this method as a means of specifically recognizing motor neurons (Figure 1 in (Mojsilovic-Petrovic et al., 2006b)). In experiments involving recombinant HSV, 1 μL of viral stock was added to 1mL of culture media 24 hours or more prior to the next manipulation. Tubes containing viruses were color coded so that the operator was blinded to the specific virus used.
The # of immunostained cells were counted in 3 randomly selected fields/coverslip and the mean value obtained. In each experiment 3+ independent coverslips were used per condition and the results presented were obtained for 4+ independent cultures and experiments.
Tissue culture cells were fixed in freshly prepared 4% paraformaldehyde in 0.1 M pH 7.4 phosphate buffer for 30 minutes prior to extensive washing in phosphate buffered saline. Overnight incubation with primary antibody was performed at room temperature and after washing, coverslips were incubated with Alexafluor conjugated secondary antibody (2 – 4 hrs.). When double labeling experiments were performed, species-specific secondary antibodies with distinct emission spectra were employed. Coverslips were washed prior to mounting in PermaFluor (Thermo Electron Corporation) and viewing on an Olympus FV300 Fluoview laser confocal microscope.
Cultures were lysed in NP-40 lysis buffer (1% NP-40, 40 mM Tris pH = 7.4, 0.15 M NaCl, 10% glycerol, 0,1% SDS, 0.1% deoxycholate + protease inhibitors and phosphatase inhibitors), sonicated, particulate matter removed by centrifugation and subjected to PAGE-SDS prior to transfer to nitrocellose. Equal amounts of protein (determined using BCA reagents from Pierce) were loaded in each lane. After blocking in 5% milk in phosphate buffer saline, membranes were incubated in primary antibody overnight, washed, incubated with secondary antibody, washed and visualized to GE Healthcare (Buckinghamshire, U.K.) chemiluminescent substrate according to the manufacturers directions. Densitometric analysis of films was obtained using TINA (Isotopenmeβgeräte, GmbH) from 4+ independent experiments, the results averaged and mean values ± S.E. formed the basis of the statistical comparisons. Quantitative data on band intensity was expressed as the fold change in comparison with values of HSV-LacZ infected cultures. The displayed western blot data are representative of the results obtained from at least 4 independent cultures. In some experiments the LI-COR Odysseus system was used for visualizing and quantifying western blot bands. Secondary antibodies were from LI-COR Biosciences (IR 800 or 680 goat anti-mouse IgG or anti-rabbit IgG). The source of primary antibodies was: (Calbiochem, Oncogene Research Products, rabbit anti- MnSOD (Stressgen Bioreagents, Victoria, British Columbia, Canada), rabbit anti- FKHRL1/FOXO3a and anti-phospho FKHRL1/FOXO3a (Thr 32) (Upstate, Lake Placid, NY), and rabbit anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA). Species-specific HRP-conjugated secondary antibodies were from Amersham (GE Healthcare, Buckinghamshire, U.K.) and Alexa 488-conjugated anti rabbit secondary antibody from Molecular Probes, Invitrogen (Eugene, OR).
Nuclear protein lysate was prepared using Sigma N-Xtract Tm kit. Briefly, cells collected from one 60 mm dish were suspended in 150 ul hypotonic lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, and 10 mM KCl). After incubating on ice for 10 minutes, 10% IGEPA CA-630 was added to final concentration of 0.6%. Vortexed vigorously for 10 seconds and centrifuged immediately at 10,000-11,000g for 30 seconds. The supernatant is cyoplasmic fraction and the pellet is nuclear fraction.
In order to identify genes that were differentially expressed in ALS we accessed the NCBI GEO database and searched the available data sets for ALS-related microarray data. Five data sets were identified and these were analyzed using the two-tailed t-test analysis with a 0.050 significance level, as provided on the GEO site, to compare the control group to the ALS group in each of the data sets. The data from these differentially expressed genes was then downloaded into a spreadsheet and then searched for genes that were determined to be controlled by FOXO as determined by Ramaswamy et. al. (Ramaswamy et al., 2002). Genes that were present in both lists were added to the Supplement Table and their direction of regulation was indicated.
B6SJL-Tg(SOD1-G93A)1Gur/J mice were obtained from Jackson labs and were bred to the F1 generation of C57Bl/6 × SJL mice. The offspring of this cross was used for all of the biochemical studies (n=4 in each experimental group). Lumbar spinal cord was obtained at P87, frozen on dry ice until use. The spinal cord segments were homogenized with a dounce in NP-40 buffer as above (10:1 volume to weight). All other manipulation of the lysates was as described tissue culture cells.
Drosophila stocks were crossed on standard cornmeal agar media at 29°C. Food was supplemented with DHT (Steraloids) and Psammaplysene A once it had cooled to <50 °C to final concentrations of 1 mM and 0.5 mM, respectively. Eye phenotypes of female flies of each condition were examined and blindly scored according to the criteria described with one scoring modification to increase sensitivity to differences in affected eye areas (Pandey et al., 2007).
Flies haploinsufficient for dFOXO were generated by mating the [GMR-GAL4, UAS-AR52Q] strain to two different null alleles – dFoxo21/Tm6B and dFoxo25/Tm6B (Junger et al., 2003), where Tm6B is the balancer chromosome. Flies containing the balancer chromosome [Tm6B/+] served as the controls for these experiments.
The following C. elegans strains were obtained from the C. elegans Genetic Center (biosci.umn.edu/CGC) or constructed by us: •glt-3: ZB1096 glt-3 (bz34) IV; nuIs5: KP742 [glr-1::gfp; glr-1::Gs (Q227L) V; lin-15(+)]; •glt-3; nuIs5: ZB1102 glt-3 (bz34) IV; nuIs5 V; age-1; •glt-3;nuIs5 Strain ZB1102 glt-3 (bz34) IV; nuIs5 V was crossed with strain BE13 sqt-1(sc13) II. sqt-1 was then kicked out and replaced by age-1 using strain TJ1052 age-1(hx546) II. Pmyo-3:daf-16::gfp Strain ZB2283 was produced by Jian Xue and Carolina Ibanez-Ventoso by making transgenic animals carrying an extra-chromosomal array containing Pmyo-3:daf-16:gfp.
For PA treatment, we soaked the diluted drug into standard nematode NGM culture plates. We transferred freshly growing nematode cultures to these plates and allowed them to grow for 2 days before assessing the effect. To determine the ratio of nuclear vs. cytoplasmic DAF-16 levels we used a strain transgenic for Pmyo-3:daf-16::gfp, where the DAF-16::GFP reporter is expressed in body wall muscle cells. We compared the intensity of GFP labeling in the nucleus and adjacent cytoplasm using NIH-Image. We monitored the effect of PA or age-1 on nematode excitotoxicity by measuring the extent of neurodegeneration in •glt-3; nuIs5 animals ± PA or ± age-1 mutation. We observed free-moving animals with an inverted scope under Nomarski DIC optics with no anesthetics. Swollen cells in the nerve-ring region were counted as head necrotic figures indicative of neurodegeneration.
Pairwise comparisons employed two-tailed Students's t-test and when three or more groups were compared we used Analysis of Variance (ANOVA) and post hoc analysis with significance set at p< 0.05.
We thank Dr. David Borchelt for the gift of the anti-human SOD rabbit serum and Ernst Hafen and Julia Lüdke (Institute of Molecular Systems Biology, Zurich) for the Foxo21 and Foxo25 fly lines. This work was supported by the U.S. Public Health Service (NS34435, MD, CA24487, JC; NS32214, DEM; NS053825, JPT and NS52325, RGK) and the ALS and Muscular Dystrophy Associations.
The authors state that they have no conflicts of interest related to this work.