Fragile X syndrome (FXS) is a neurodevelopmental disorder with a wide spectrum of cognitive and behavioral problems [for review, see (Krueger and Bear 2011)]. Loss of fragile X mental retardation 1 (Fmr1) gene expression is an established cause of FXS (Krueger and Bear 2011). The expansion of CGG repeats (> 200) in 5′ untranslated regions of Fmr1 gene results in hypermethylation, transcriptional silencing, and loss of fragile X mental retardation protein (FMRP) (Chakrabarti and Davies 1997).
FMRP function is thought to be mediated largely by its ability to bind mRNAs through two well-known RNA-binding domains, like K-homology domains, and RGG (arg-gly-gly triplet) box (Siomi et al. 1993). Through these domains, FMRP recognizes, regulates (mainly represses) translation, and transports specific mRNAs (Brown et al. 2001; Zalfa et al. 2007; Bassell and Warren 2008; Dictenberg et al. 2008). In neurons, FMRP may modulate expression of mRNAs by controlling recognition, export, translational efficiency, and stability of target mRNAs (Darnell et al. 2001; De Rubeis and Bagni 2010). Meanwhile, it is also known that FMRP has a nuclear localization signal (NLS) and a nuclear export signal (Eberhart et al. 1996), suggesting that it shuttles between the nucleus and cytoplasm. In fact, FMRP is found in both nucleus and cytoplasm, an indication that it may have several independent functions within the cell such as the regulation of cell division, growth, and survival (Siomi et al. 1993). Among the various mRNAs which might be regulated by FMRP, some are related to the regulation of cell survival and death. These include components of Bcl-2 family-like Bcl-2-interacting protein (Bnip) and Bcl-2-interacting killer as well as signaling molecules such as NF-kB, PKC, and Mitogen-activated protein kinase (Brown et al. 2001; Chen et al. 2003).
Many studies have identified Ras-phosphatidylinositol 3-kinase (PI3K)- Protein Kinase B (Akt) as a major cell survival signaling pathway (Engelman et al. 2006; Chalhoub et al. 2009; Wagner-Golbs and Luhmann 2012), which makes the pathway an attractive target for therapeutics against many forms of cancer with misregulated cellular apoptosis (Vivanco and Sawyers 2002). Akt is a serine/threonine protein kinase that plays a key role in multiple cellular processes such as cell proliferation, apoptosis, and transcription (Datta et al. 1997). Among these various functions, Akt indirectly activates NF-kB transactivation by dissociation of phosphorylated Ik-B kinase, resulting in transcription of pro-survival genes like B-cell lymphoma-extra large (Bcl-xL) and trophic factors (Brunet et al. 2001). In addition, trophic events alleviate excitotoxic and ischemic injury by activating PI3K-dependent Akt activation. Collectively, Akt signaling pathway seems to be one of the major mediators or determinants of cellular survival or death.
Interestingly, abnormal Ras-PI3K-Akt signaling cascades were reported in Fmr1 knockout animals (Hu et al. 2008). Although both Ras–MEK–ERK1/2 Mitogen-activated protein kinase cascades and Ras-PI3K–Akt cascades were normal in wild-type and Fmr1 knockout animals in basal condition, the stimulation-induced activation of PI3K–Akt was weakened and abnormal in Fmr1 knockout animals (Hu et al. 2008). Interestingly, standardized incidence ratio of cancer was reduced to 0.28 (95.0% ± 0.8) compared to control in a study of Dutch FXS patients (Schultz-Pedersen et al. 2001). These results suggest that down-regulation of FMRP may increase cell death via abnormal Ras-PI3K-Akt signaling in stimulated condition, which may misregulate downstream targets such as Bcl-xL. In this study, we hypothesized that FMRP may control cell survival and death in neurons via the regulation PI3K-Akt pathway and investigated the possibility using in vitro glutamate stimulation and in vivo transient focal ischemia (middle cerebral artery occlusion, MCAO) paradigm.
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FMRP has been implicated in various neurological diseases including FXS, autism, epilepsy, and attention deficit/hyperactivity disorder. As such, research efforts have been focused on understanding the function of FMRP during development and the regulation of synaptic protein expression. However, even in this relatively intensely studied field, the exact regulatory mechanisms and functions of FMRP, especially in the context of normal and stressed condition, have yet to be fully elucidated.
In the work presented here, we described a pro-survival function of FMRP in primary cortical neurons. Using loss- and gain- of functional analysis, we showed that FMRP promoted cell survival under basal states and protects against cell death following toxic stimuli. We employed two common methods to induce neuronal stress, excessive glutamate stimulation in vitro and ischemia in vivo. In both cases, cell death was amplified in the absence of FMRP and tempered following over-expression of FMRP.
After bath application of [(S)-3,5-Dihydroxyphenylglycine], which is a metabotropic glutamate receptor 1/5 agonist, the level of FMRP was biphasically regulated by ubiquitin-dependent proteolysis system and translational control of FMRP as well as other regulator proteins (Hou et al. 2006; Zhao et al. 2011). Similar biphasic regulation of FMRP level was also observed in our study, in this case with initial increase of FMRP until 1 h, followed by down-regulation to basal level, although it remains to be determined how it can be reconciled with the observed changes in the activity of PI3K-Akt pathway. The rapid induction of FMRP in our study may also suggest translational control mechanism of FMRP expression at least in the early stage of cellular stress. Actually, glutamate-induced rapid up-regulation of FMRP in cultured primary neuron was not affected by the pre-treatment of a transcriptional inhibitor actinomycin D, however, the pre-treatment of a translational inhibitor anisomycin significantly reduced FMRP expression in control and glutamate-stimulated cells (our unpublished results). Similarly, other researchers reported that mGluR-dependent rapid FMRP expression is regulated by translational control (Weiler et al. 1997; Narayanan et al. 2008), which might be regulated by activation of PI3K-Akt-mTOR pathway (Hou and Klann 2004).
Previously, Khalil et al. showed a modest but significant decrease in HeLa cell proliferation after Fmr1 siRNA-induced knockdown of FMRP (Khalil et al. 2008) and we reported a decrease in HeLa cell viability following Fmr1 siRNA in an etoposide-stimulated cell toxicity model (Jeon et al. 2011b). Similarly, normal hippocampal neuronal viability and development was reduced in Fmr1 knockout mice compared with wild type (Jacobs and Doering 2010), and neural stem cells from Fmr1 knockout mice showed increased cell death (Castren et al. 2005; Castren 2006). Taken together with our results, these data strongly implicate an additional role of FMRP as a pro-survival protein.
Regarding the pro-survival role, the possible involvement of FMRP in the Ras pathway was first described in lymphocyte of FXS patients, which showed decreased expression of Ras-GTPase-Activating protein SH3-domain-binding protein, an effector of Ras signal transduction pathway (Zhong et al. 1999). Ras-GTPase-Activating protein SH3-domain-binding protein is expressed in brain (Atlas et al. 2004) and affects proliferation, differentiation, and survival of cell (Zhang and Shao 2010). Hu et al. reported that Ras-PI3K-Akt signaling is muted in FMRP knockout animals, suggesting the possibility that FMRP regulates Ras-PI3K signaling pathway (Hu et al. 2008). FXS patients showed lower cancer incidence compared with normal subjects (Schultz-Pedersen et al. 2001), and it is speculated that altered cell death regulatory mechanism, possibly via misregulated Ras-PI3K-Akt signaling in FXS patients, might underlie the lower cancer incidence (Hu et al. 2008).
In this study, FMRP expression was regulated by PI3K or Akt activation. In turn, increased level of FMRP seems essential for the up-regulation of Akt activity as evidenced by shRNA and over-expression studies, which forms a positive feedback loop. It is unclear how transient increase in FMRP may lead to activation of PI3K in our condition. In Fmr1 knockout hippocampal CA1 cells, defective histamine or acetylcholine-induced activation of PI3K-Akt pathway has been reported, albeit with the increased basal Ras activity (Hu et al. 2008). Interestingly, the basal level of Ras-dependent phosphorylation of Akt between WT and Fmr1 knockout mice was not different, which suggests that the expression of FMRP might be important in the stimulus-dependent but not basal level of activation of PI3K, at least to the downstream of Ras pathway (Hu et al. 2008). Similarly, it has been reported that [(S)-3,5-Dihydroxyphenylglycine]-induced phosphorylation of the downstream effectors of PI3K, such as phosphoinositide-dependent protein kinase 1, Akt, mTOR, and ribosomal p70S6 kinase, but not the basal level of phosporylation is impaired in Fmr1 knockout mice (Ronesi and Huber 2008), which may result from the reduced mGluR-long Homer interaction in Fmr1 knockout mice. Although other researchers reported up-regulation of PI3K activity in Fmr1 knockout mice (Gross et al. 2010; Sharma et al. 2010), these results may suggest a hypothesis that transient increase in FMRP may increase stimulus-dependent coupling of PI3K to upstream regulators by mechanisms hitherto unknown. Recently, it was reported that PTEN, a PI3K inhibitor and its mRNA is a putative target of FMRP, was dephosphorylated in Fmr1 knockout mice, although it is not sufficient to modulate PI3K activity (Sharma et al. 2010). Whether transient induction of FMRP in our condition may affect PTEN activity or expression to sufficient extent to modulate PI3K activity remains to be determined. Another candidate molecule which might be mediating the regulation of PI3K-Akt pathway through FMRP is protein phosphatase 2A (PP2A). The mRNA for PP2A has been suggested as a target of FMRP (Waggoner and Liebhaber 2003) and FMRP inhibited the translation of PP2A to act as a negative regulator of PP2A (Castets et al. 2005). Considering the role of PP2A in the inhibitory regulation of Akt activity among others (see a recent review by Bononi et al. 2011), it is also possible that the changes in the level of FMRP in our condition may exert its effects on PI3K-Akt pathway by modulating the level and activity of PP2A, which again needs experimental evidences in the future.
In contrast to our results, other researchers reported excess PI3K activation in neurons from Fmr1 knockout mice (Gross et al. 2010; Sharma et al. 2010). At present, the reason underlying those different results are not clear. Because we used shRNA instead of cells derived from knockout animals, the most evident difference would be the transient and acute nature of the FMRP down-regulation in our condition as compared with knockout animals. In situations where long-term changes in FMRP expression happen (knockout mice and human patients), compensatory responses may occur to minimize the deleterious effects to cells. Second, although the exact types of subpopulation of cells more vulnerable to FMRP loss is not obvious from this study, susceptible cells might be lost already in FMRP knockout mice during development, which may make it difficult to observe massive cell death in FMRP knockout mice. Investigating such cell types, for example, neural progenitor cells in specified loci and developmental stage, would be an interesting topic.
Recently, FMRP has been associated with the regulation of cellular mitosis in neural stem cells. Luo et al. showed almost 52.0% increase in Bromodeoxyuridine (BrdU) (S phase marker) positive neural stem/progenitor cells in Fmr1 knockout animals and suggested the mechanism by translational control of cyclin D1 and cyclin-dependent kinase 4 (CDK4) (Luo et al. 2010; Callan and Zarnescu 2011; Guo et al. 2011), both of which are well-known partners of cell cycle progression enhancers. In contrast to above results, our results suggest that knockdown of FMRP in cultured neuron decreases survival of neurons. The innate property of neuron versus neural stem cell may explain the discrepancy. Unlike to neural stem cell, the overt activation of cell cycle progression in neuron, which is post-mitotic, has been implicated in ectopic cell death in pathological conditions including AD, PD, and stroke [for a review, see (Lopes and Agostinho 2011)]. Neurological insults such as glutamate excitotoxicity and stroke induce oxidative stress, which result in overt activation of cyclin-dependent kinase 5 (CDK5) and CDK4 in neurons. CDK4 over-expression (activation) led neurons to re-enter the cell cycle (from G1 to S and G2 phase). However, the re-entry into cell cycle does not reach the M phase and initiates degenerative process (apoptosis) by activating caspase 3 and pro-apoptotic factors of Bcl-2 (Lopes and Agostinho 2011). Therefore, the same molecular events mediated by down-regulation of FMRP, i.e. over-activation CDK5/4 may mediate mitosis in neural stem cell, but susceptibility to cell death in neuron. Interestingly, kinetic study of BrdU-positive cells in brain revealed that the survival of BrdU-labeled cells over 4-weeks span was lower in FMRP knockout mice suggesting that FMRP deficiency might reduce cell survival of young neurons (Luo et al. 2010), which is consistent with our results. Taken together, these results suggest that the loss of FMRP expression may reduce cell survival of (young) neuron, although it may induce cell proliferation of neural stem cells.
Even though the primary localization of FMRP is cytoplasm in immunocytochemical staining or western blot with biochemically fractionated samples (Devys et al. 1993; Feng et al. 1997), it contains a functional, non-classical NLS near its N terminus (Eberhart et al. 1996; Sittler et al. 1996; Bardoni et al. 1997) with occasional nuclear localization (Feng et al. 1997; Zhang et al. 2007; Kim et al. 2009). FMRP-GFP- or eGFP-FMRP-transfected cells showed strong nuclear localization with bound mRNAs (De Diego Otero et al. 2002) and shuttles between cytoplasm and nucleus. In an experiment using a series of mutant with NLS and nuclear export signal, it has been suggested that FMRP primarily binds target mRNAs in nucleus although it is effectively transported out by interaction with RNAs and Tap/NXF1, a bulk mRNA exporter (Kim et al. 2009). Silencing of Tap/NXF1 increased nuclear localization of eGFP-FMRP (Kim et al. 2009). These results raise interesting possibility that either retention of RNAs in the nucleus or modulation of FMRP interacting proteins such as Tap/NXF1 or nuclear FMRP-interacting protein 1 (Bardoni et al. 2003) may retain FMRP in nucleus. Recently, nuclear retention and survival role of other RNA-binding proteins has also been suggested. RNA-binding proteins, cytoplasmic polyadenylation element binding (CPEB) 1, 3, and 4, are accumulated in nucleus of neurons after treatment with ionotropic glutamate receptor agonists in intracellular calcium and alpha-CAMKII-dependent manner (Kan et al. 2010). Among those CPEB isotypes, CPEB4 has been suggested to be important in cell survival during focal ischemia in vivo and oxygen–glucose deprivation in vitro (Kan et al. 2010). With these several intriguing possibilities including the role of PI3K/Akt pathway in the regulation of nuclear localization of FMRP in mind, we are actively investigating the mechanism and role of nuclear FMRP translocation in the regulation of neuronal survival.
In conclusion, our experimental results suggest that physiological regulation of FMRP may enhance cellular survival against neurotoxic stimulations such as ischemic stroke. Investigating whether endogenous expression of FMRP may perform neuroprotective roles against other neurodegenerative disorders might provide a better understanding and therapeutic targeting of these diseases.
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Figure1. Measurement of neuronal cell death.
Figure2. Role of PI3K-Akt signaling pathways on glutamate-induced FMRP expression.
Figure3. Knock-down of FMRP expression sensitizes rat primary cortical neuron to glutamate-induced cell death in culture.
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