Altered mRNA transport, docking, and protein translation in neurons lacking fragile X mental retardation protein

Abstract

Fragile X syndrome is caused by the absence of functional fragile X mental retardation protein (FMRP), an RNA binding protein. The molecular mechanism of aberrant protein synthesis in fmr1 KO mice is closely associated with the role of FMRP in mRNA transport, delivery, and local protein synthesis. We show that GFP-labeled Fmr1 and CaMKIIα mRNAs undergo decelerated motion at 0-40 min after group I mGluR stimulation, and later recover at 40-60 min. Then we investigate targeting of mRNAs associated with FMRP after neuronal stimulation. We find that FMRP is synthesized closely adjacent to stimulated mGluR5 receptors. Moreover, in WT neurons, CaMKIIα mRNA can be delivered and translated in dendritic spines within 10 min in response to group I mGluR stimulation, whereas KO neurons fail to show this response. These data suggest that FMRP can mediate spatial mRNA delivery for local protein synthesis in response to synaptic stimulation.fragile X syndrome | dendritic mRNA targeting | local translation F ragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function of the FMR1 gene, which encodes fragile X mental retardation protein (FMRP) (1). FXS affects 1 in 4,000 males and 1 in 6,000 females on average and is characterized by hyperactivity, attention deficits, autistic-like behaviors, and seizures (2). Dendritic spine morphology in the cerebral cortex of FXS patients and in the fmr1 KO mouse model shows more immature long thin spines than mature stubby, mushroom-shaped spines (3). Furthermore, group I mGluR-dependent long-term depression in the hippocampus is exaggerated in the fmr1 KO model (4). These findings suggest that FMRP functions in synaptic development and plasticity.Activity-dependent local translation is a fundamental mechanism underlying synaptic plasticity (5, 6). Inhibition of protein synthesis attenuates specific types of long-term plasticity (7,8). Morphological changes in dendritic spines can be blocked by protein synthesis inhibitors (9). In the fmr1 KO model, it has been shown that aberrant synthesis of individual proteins such as CaMKIIα, PSD-95, and MAP1b, upon group I mGluR stimulation, is associated with defective long-term plasticity (10-12). Here we have studied specific molecular mechanisms to elucidate aberrant localized translation in the fmr1 KO model. The molecular basis of FMRP's role in translation-dependent plasticity remains unclear despite extensive study. FMRP is a ribosome-associated RNA binding protein with selective affinity (13,14). Upon neuronal stimulation, FMRP may regulate protein levels by mediating translational regulation and mRNA trafficking (11, 15). FMRP, mRNA, and other RNA binding proteins can form ribonucleoprotein (RNP) or granule structures and couple with motor proteins to be transported in dendrites (16-18). Dendritic transport of FMRP and associated mRNAs, such as Fmr1, CaMKIIα, and MAP1b, are regulated by group I mGluR signaling (15, 19). It is not yet fully understood how and when mRNA is delivered to th...

Integrated transcriptome analysis of human iPS cells derived from a fragile X syndrome patient during neuronal differentiation

“…In the nervous system, loss of FMRP elevates the protein synthesis in the brain (Laggerbauer et al, 2001;Qin et al, 2005), weakens the synaptic strength and affects dendritic spines (Cruz-Martin et al, 2010;Pan et al, 2010;Pfeiffer and Huber, 2007), which therefore affects many different pathways, e.g., impairing metabotropic glutamate receptor (mGluR)-mediated signaling (Bear et al, 2004) , reducing GABAergic transmission (D'Hulst and Kooy, 2007) and enhancing mTOR signaling (Sharma et al, 2010). The function of FMRP in cytoplasm was well characterized as a selective RNA-binding protein that negatively regulates the translation of the target mRNAs for presynaptic, postsynaptic proteins, and interestingly, many transcriptional factors (Ascano et al, 2012;Auerbach et al, 2011;Darnell et al, 2011;Kao et al, 2010;Lu et al, 2004;Muddashetty et al, 2007;Osterweil et al, 2010;Pfeiffer and Huber, 2007). FMRP also comprises the nuclear export signal (NES) and nuclear localization signal (NLS) domains and shuttles between the nucleus and cytoplasm (Eberhart et al, 1996;Feng et al, 1997;Sittler et al, 1996).…”

Fmr1 deficiency promotes age-dependent alterations in the cortical synaptic proteome

“…Moreover, because it is possible that FMRP may promote synaptic expression of some of its targets through effects on mRNA stability or transport (4,5), it cannot be reliably inferred that a direct target of FMRP is up or downregulated in FXS. Such complexities can be captured only by direct quantification of changes at the protein level.…”

“…to capture secondary effects of FMRP loss on the proteome, and they may render both false-positives and false-negatives resulting, respectively, from potential "bystander" roles of FMRP and effects that occur outside the context of polysomes (e.g., mRNA stability and transport) (4,5). Moreover, because it is possible that FMRP may promote synaptic expression of some of its targets through effects on mRNA stability or transport (4,5), it cannot be reliably inferred that a direct target of FMRP is up or downregulated in FXS.…”

“…In the vast majority of cases, FXS is caused by an mRNA-dependent epigenetic silencing of the Fmr1 (fragile X mental retardation 1) gene (2), which occurs secondarily to a CGG repeat expansion in the 5′ UTR region of Fmr1 (3) and results in absence of the encoded protein FMRP. FMRP is an RNA-binding protein that regulates several aspects of mRNA translation (1), transport (4), and stability (5) in neurons. Substantial evidence indicates that FMRP is particularly critical as a suppressor of activity-dependent mRNA translation at glutamatergic synapses (6,7) and that loss of this function results in abnormalities in dendritic spine shape and several forms of long-term synaptic plasticity (8,9).…”

The molecular basis of cognitive deficits in pervasive developmental disorders: Table 1.

“…The most extensively studied of this type of PDD is fragile X syndrome (FXS), which is caused by an uncontrolled expansion of CGG repeats at the 5 ′ end of the Fmr1 gene, causing promoter hypermethylation and subsequent loss of expression of fragile x mental retardation protein (FMRP). FMRP mediates different aspects of RNA metabolism, including trafficking of RNP particles, translation of specific mRNA transcripts via regulation of translation initiation and elongation, and targeted degradation via the RISC complex (Jin et al 2004;Park et al 2008;Kao et al 2010;Melko and Bardoni 2010). FXS model mice, where the Fmr1 gene has been deleted, have been exhaustively studied and shown to display the "triad of deficits" features associated with PDD (Gross et al 2012).…”