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 (). 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 (). 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.
Figure 1 Triple-mutant FOXO3a is retained in the nucleus and protects against excitotoxic and proteotoxic insults. Panel A. Mixed spinal cord neurons were transfected using Lipofectamine 2000 (Invitrogen) with HA-tagged wild type FOXO3a (WT-FOXO3a) or triple mutant (more ...)
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) (). 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 ().
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 (). 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 (). 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) (). 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 () (Schroeder et al., 2005
Figure 2 Psammaplysene A (PA) drives FOXO3a into the neuronal nucleus and protects against the proteotoxicity of mutant SOD and mutant p150glued. Panel A. The chemical structure of PA. Panel B. 14 DIV spinal cord cultures were treated with PA or vehicle for 24 (more ...)
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 (). We next biochemically isolated nuclei from spinal cord cultures treated with PA or vehicle (). 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 (). 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 (). 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) (). 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 (). 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
) (). 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.
Figure 3 Effects of PA on the distribution of WT and mutant SOD in vitro and biochemical consequences of WT and mutant SOD in vitro and in vivo. Panel A. Mixed spinal cord cultures infected with HSV-WT-SOD or HSV-G87R-SOD were treated with PA or vehicle overnight (more ...)
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 (). 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 (). 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 (). 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 (). 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) (). 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 (). 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.
Figure 4 PA suppresses degeneration in a Drosophila model of SBMA in a dFOXO-dependent manner. Right panel of each diptych shows higher magnification of the posterior margin of the eye, where degeneration is concentrated. (a, b) Flies expressing polyglutamine-expanded (more ...)
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
) (). 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 (). 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 (). 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
) 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 (). 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 (). 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 5 PA treatment or reducing the activity of IGFR signaling pathway alleviate nematode excitotoxicity. Panel A. PA has a dose-dependent neuroprotective effect on nematode excitotoxicity: •glt-3;nuIs5 double mutants nematodes were cultured in presence (more ...)
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) ().
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) (). 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.