BDNF Induced Translation of Limk1 in Developing Neurons Regulates Dendrite Growth by Fine-Tuning Cofilin1 Activity.

Dendritic growth and branching are highly regulated processes and are essential for establishing proper neuronal connectivity. There is a critical phase of early dendrite development when these are heavily regulated by external cues such as trophic factors. Brain-derived neurotrophic factor (BDNF) is a major trophic factor known to enhance dendrite growth in cortical neurons, but the molecular underpinnings of this response are not completely understood. We have identified that BDNF induced translational regulation is an important mechanism governing dendrite development in cultured rat cortical neurons. We show that BDNF treatment for 1 h in young neurons leads to translational up-regulation of an important actin regulatory protein LIM domain kinase 1 (Limk1), increasing its level locally in the dendrites. Limk1 is a member of serine/threonine (Ser/Thr) family kinases downstream of the Rho-GTPase pathway. BDNF induced increase in Limk1 levels leads to increased phosphorylation of its target protein cofilin1. We observed that these changes are maintained for long durations of up to 48 h and are mediating increase in number of primary dendrites and total dendrite length. Thus, we show that BDNF induced protein synthesis leads to fine-tuning of the actin cytoskeletal reassembly and thereby mediate dendrite development.

Genetic and acute CPEB1 depletion ameliorate fragile X pathophysiology.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.

Regulation of zipcode binding protein 1 transport dynamics in axons by myosin Va.

Directed transport of the mRNA binding protein, zipcode binding protein1 (ZBP1), into developing axons is believed to play an important role in mRNA localization and local protein synthesis. The role of molecular motors in this process is unclear. We elucidated a role for myosin Va (MyoVa) to modulate the axonal localization and transport of ZBP1 in axons. Using cultured rat hippocampal neurons, ZBP1 colocalized with MyoVa in axons and growth cones. Interaction of MyoVa with ZBP1 was evident by coimmunoprecipitation of endogenous and overexpressed proteins. Inhibition of MyoVa function with the globular tail domain (GTD) of MyoVa protein or short hairpin RNA led to an accumulation of ZBP1 in axons. Live cell imaging of mCherryZBP1 in neurons expressing GTD showed an increase in the number of motile particles, run length, and stimulated anterograde moving ZBP1 particles, suggesting that MyoVa controls availability of ZBP1 for microtubule-dependent transport. These findings suggest a novel regulatory role for MyoVa in the transport of ZBP1 within axons.

Dephosphorylation-induced ubiquitination and degradation of FMRP in dendrites: a role in immediate early mGluR-stimulated translation.

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation of FMRP and target mRNAs from miRNA-induced silencing complexes. Here, we report the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation of FMRP in dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95 mRNA, required both PP2A and UPS. Thus, control of FMRP levels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis.

Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation, and mGluR signaling.

The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.