Fragile X related protein 1 clusters with ribosomes and messenger RNAs at a subset of dendritic spines in the mouse hippocampus

The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.     

Acute BDNF Treatment Upregulates GluR1-SAP97 and GluR2-GRIP1 Interactions: Implications for Sustained AMPA Receptor Expression

Brain-derived neurotrophic factor (BDNF) plays several prominent roles in synaptic plasticity and in learning and memory formation. Reduced BDNF levels and altered BDNF signaling have been reported in several brain diseases and behavioral disorders, which also exhibit reduced levels of AMPAr subunits. BDNF treatment acutely regulates AMPA receptor expression and function, including synaptic AMPAr subunit trafficking, and implicates several well defined signaling molecules that are required to elicit long term potentiation and depression (LTP and LTD, respectively). Long term encoding of synaptic events, as in long term memory formation, requires AMPAr stabilization and maintenance. However, factors regulating AMPAr stabilization in neuronal cell membranes and synaptic sites are not well characterized. In this study, we examine the effects of acute BDNF treatment on levels of AMPAr-associated scaffolding proteins and on AMPAr subunit-scaffolding protein interactions. We also examine the effects of BDNF-dependent enhanced interactions between AMPAr subunits with their specific scaffolding proteins on the accumulation of both types of proteins. Our results show that acute BDNF treatment upregulates the interactions between AMPAr subunits (GluR1 and GluR2) with their scaffold proteins SAP97 and GRIP1, respectively, leading to prolonged increased accumulation of both categories of proteins, albeit with distinct mechanisms for GluR1 and GluR2. Our findings reveal a new role for BDNF in the long term maintenance of AMPA receptor subunits and associated scaffolding proteins at synapses and further support the role of BDNF as a key regulator of synaptic consolidation. These results have potential implications for recent findings implicating BDNF and AMPAr subunits in various brain diseases and behavioral disorders.     

The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP

Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.     

Brain-derived neurotrophic factor-tropomyosin-related kinase B signaling contributes to activity-dependent changes in synaptic proteins

The ability of synapses to undergo changes in structure and function in response to alterations of neuronal activity is an essential property of neural circuits. One way that this is achieved is through global changes in the molecular composition of the synapse; however, it is not clear how these changes are coupled to the dynamics of neuronal activity. Here we found that, in cultured rat cortical neurons, bidirectional changes of neuronal activity led to corresponding alterations in the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor tropomyosin-related kinase B (TrkB), as well as in the level of synaptic proteins. Exogenous BDNF reversed changes in synaptic proteins induced by chronic activity blockade, while inhibiting Trk kinase activity or depleting endogenous BDNF abolished the concentration changes induced by chronic activity elevation. Both tetrodotoxin and bicuculline had significant, but opposite, effects on synaptic protein ubiquitination in a time-dependent manner. Furthermore, exogenous BDNF was sufficient to increase ubiquitination of synaptic proteins, whereas scavenging endogenous BDNF or inhibiting Trk kinase activity prevented the ubiquitination of synaptic proteins induced by chronic elevation of neuronal activity. Inhibiting the proteasome or blocking protein polyubiquitination mimicked the effect of tetrodotoxin on the levels of synaptic proteins and canceled the effects of BDNF. Our study indicates that BDNF-TrkB signaling acts upstream of the ubiquitin proteasome system, linking neuronal activity to protein turnover at the synapse.     

Role for brain-derived neurotrophic factor in learning and memory

In addition to its actions on neuronal survival and differentiation, brain-derived neurotrophic factor (BDNF) has a role in the regulation of synaptic strength. Long-term potentiation, a form of synaptic plasticity, is markedly impaired in BDNF mutant mice, but the changes were restored by the re-expression of BDNF. BDNF also influences the development of patterned connections and the growth and complexity of dendrites in the cerebral cortex. These results suggest a role for BDNF in learning and memory processes, since memory acquisition is considered to involve both short-term changes in electrical properties and long-term structural alterations in synapses. Memory acquisition is associated with an increase in BDNF mRNA and TrkB receptor activation in specific brain areas. Moreover, the pharmacologic and genetic deprivation of BDNF or its receptor TrkB results in severe impairment of learning and memory in mice, rats and chicks. The effect of BDNF on learning and memory may be linked to the modulation of NMDA and non-NMDA receptor functions as well as the expression of synaptic proteins required for exocytosis. Activation of the mitogen-associated protein kinase and/or phosphatidylinositol 3-kinase signaling pathways may be involved in BDNF-dependent learning and memory formation. It is concluded that BDNF/TrkB signaling plays an important role in learning and memory.     

Activity-dependent and hormonal regulation of neurotrophin mRNA levels in brain–implications for neuronal plasticity

The neurotrophins exhibit neurotrophic effects on specific, partially overlapping populations of neurons both in the peripheral and the central nervous system (CNS). In the periphery, they are synthesized by a variety of nonneuronal cells, and their synthesis seems to be independent of the neuronal input. In contrast, in the CNS all neurotrophins are expressed under physiological conditions primarily by neurons. The production of NGF and BDNF is controlled by neuronal activity: up-regulation by glutamate and acetylcholine, down-regulation by gamma-aminobutyric acid. In contrast, NT-3 regulation is independent of neuronal activity, but it is up-regulated by thyroid hormones and BDNF. The latter observation suggests that NT-3 might be controlled indirectly by neuronal activity via BDNF. In peripheral nonneuronal tissues, glucocorticoid hormones down-regulate NGF mRNA levels both in vitro and in vivo. In contrast, in the CNS, neuronal production of NGF is enhanced by glucocorticoids. The rapid regulation of NGF and BDNF by subtle physiological stimuli together with the recent demonstration that the neurotrophin release neurotransmitters such as acetylcholine opens up interesting perspectives for the function of neurotrophins as mediators of neuronal plasticity. 1994 John Wiley & Sons, Inc.     

Lactobacillus rhamnosus GG and Bifidobacterium bifidum TMC3115 Can Affect Development of Hippocampal Neurons Cultured In Vitro in a Strain-Dependent Manner

This study examined whether Lactobacillus rhamnosus GG (LGG) and Bifidobacterium bifidum TMC3115 (TMC3115) could morphologically or physiologically influence hippocampal neuronal development in vitro. Hippocampal neurons cultured in vitro were exposed to live or heat-inactivated LGG or TMC3115 for either 6 or 24 h. Neuronal morphological changes and drebrin (DRB) and synaptophysin (SYP) protein levels were monitored using immunofluorescence. And the levels of DRB, SYP, and brain-derived neurotrophic factor (BDNF), and cAMP-response element binding protein (CREB) mRNA were detected using RT-PCR. The BDNF, CREB, and phosphorylated-CREB (P-CREB) protein levels were detected by extraction-enzyme-linked immunosorbent assay (ELISA) or Western blot assays. Heat-inactivated LGG and TMC3115 could enhance neuron viability, DRB and SYP protein levels, and BDNF mRNA level were significantly altered after exposure to the tested bacteria with 6 h or 24 h. There were no significant differences in neuronal morphology or DRB, SYP, or CREB mRNA levels among the groups following bacterial exposure. However, following exposure of live TMC3115 for 24 h, the neuronal BDNF and P-CREB protein levels were both significantly up-regulated as detected by western blot assays. These results demonstrated that LGG and TMC3115 could affect neuronal viability, along with hippocampal synaptic and functional development, in a strain-dependent manner, which may also be closely associated with the physiological and culture conditions of each strain. Up-regulated P-CREB may be one of the underlying mechanisms by which the bacteria, especially neurons following exposure of live TMC3115 for 24 h, are able to regulate neuronal BDNF protein production.

     

HuD Interacts with Bdnf mRNA and Is Essential for Activity-Induced BDNF Synthesis in Dendrites

Highly specific activity-dependent neuronal responses are necessary for modulating synapses to facilitate learning and memory. We present evidence linking a number of important processes involved in regulating synaptic plasticity, suggesting a mechanistic pathway whereby activity-dependent signaling, likely through protein kinase C (PKC)-mediated phosphorylation of HuD, can relieve basal repression of Bdnf mRNA translation in dendrites, allowing for increased TrkB signaling and synaptic remodeling. We demonstrate that the neuronal ELAV family of RNA binding proteins associates in vivo with several Bdnf mRNA isoforms present in the adult brain in an activity-dependent manner, and that one member, HuD, interacts directly with sequences in the long Bdnf 3'' untranslated region (3''UTR) and co-localizes with Bdnf mRNA in dendrites of hippocampal neurons. Activation of PKC leads to increased dendritic translation of mRNAs containing the long Bdnf 3''UTR, a process that is dependent on the presence of HuD and its phosphorylation at threonine residues 149 and/or 165. Thus, we found a direct effect of HuD on regulating translation of dendritic Bdnf mRNAs to mediate local and activity-dependent increases in dendritic BDNF synthesis.     

Localized synaptic potentiation by BDNF requires local protein synthesis in the developing axon

Brain-derived neurotrophic factor (BDNF) is known to promote neuronal survival, guide axonal pathfinding, and participate in activity-dependent synaptic plasticity. In Xenopus nerve-muscle cultures, localized contact of a single BDNF-coated bead with the presynaptic axon resulted in potentiation of transmitter secretion at the developing synapses, but only when the bead was placed within 60 microm from the synapse. The localized potentiation induced by BDNF is accompanied by a persistent local elevation of [Ca(2+)](i) in the axon and requires constitutive presynaptic protein translation, even for axons severed from the cell body. Thus, presynaptic local TrkB signaling and protein synthesis allow a localized source of BDNF to potentiate transmitter secretion from nearby synapses, a property suited for spatially restricted synaptic modification by neurotrophins.     

Brain-derived neurotrophic factor induces cell surface expression of short-form tenascin R complex in hippocampal postsynapses

Brain-derived neurotrophic factor (BDNF) is involved in hippocampal functions such as learning and memory and it plays a crucial role in regulating synaptic plasticity. To investigate potential mechanisms by which BDNF participates in neuronal communication through postsynaptic membrane proteins, we generated monoclonal antibodies against the synaptoneurosomal particulate fraction of mouse brain. One of the monoclonal antibodies, termed mAb#27, was found to be useful for analyzing BDNF-induced externalization of synaptoneurosomal membrane proteins of the hippocampus. In dissociated neuronal cultures, BDNF stimulation increased mAb#27 immunoprecipitates of biotin-labeled proteins with apparent masses, 55kDa, 80kDa, 100kDa, 130kDa, 140kDa and 160kDa. The mAb#27 recognition molecules were located in specific hippocampal regions of the brain and at postsynaptic sites in cultured cells. Proteomic studies of the mAb#27 immunocomplex identified newly derived short forms of tenascin R (TNR) as the mAb#27 recognition molecule. Contactin 1, prostaglandin regulatory-like protein and GABA A receptor subunit beta3 were identified as TNR-associated proteins. These proteins were recruited to mAb#27 when BDNF was applied to cells in culture. Each molecules identified in the present study contributes to the postsynaptic plasticity or the active cycle of cellular vesicle membranes. The formation of the TNR complex may serve as an underlying basis for synaptic plasticity in the hippocampus. Our results demonstrate that BDNF plays a role in external molecular dynamics and is likely to regulate synaptic functions such as the enhancement of neuronal excitability through GABAergic neurons.     

mRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure

The neuronal Na(+)-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200μm from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.     

Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo: evidence for compartment-specific translation control

Protein synthesis underlying activity-dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor-2 (eEF2), during long-term potentiation (LTP) induced by local infusion of brain-derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF-induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked BDNF-LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho-eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of alpha-calcium/calmodulin-dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK-dependent regulation of the initiation and elongation steps of protein synthesis during BDNF-LTP in vivo. Furthermore, the results suggest a compartment-specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non-synaptic sites.     

A novel trimeric peptide, Neuropep-1-stimulating brain-derived neurotrophic factor expression in rat brain improves spatial learning and memory as measured by the Y-maze and Morris water maze

Abundant studies have shown possible links between low levels of brain-derived neurotrophic factor (BDNF) and neurological diseases such as Alzheimer's disease, Parkinson's disease, and depression, as well as stress and anxiety; therefore, BDNF could be a therapeutic target for neurological disorders. In the present study, a positional scanning-synthetic peptide combinatorial library was utilized to identify a peptide modulator of BDNF expression in the hippocampal neuronal cell line, H19-7. A novel tripeptide (Neuropep-1) induced a significant increase of BDNF mRNA and protein levels in H19-7 cells. Pre-treatment of TrkB inhibitor (K252a) did not block Neuropep-1-induced BDNF up-regulation. These results indicate that Neuropep-1 may up-regulate BDNF expression that might be independent of the TrkB receptor pathway. Tail vein injection of Neuropep-1 significantly up-regulated BDNF expression, TrkB phosphorylation, and its downstream signals including activation of Akt, ERK, and cAMP response element binding in the rat hippocampus. To evaluate improvement of spatial learning and memory (SLM) by Neuropep-1-induced BDNF up-regulation, the Y-maze and Morris water maze tests were performed. These results showed Neuropep-1 injection improved SLM performance with increase of BDNF and TrkB expression, activation of TrkB downstream signals in rat hippocampus compared with the control group. However, phosphorylation levels of TrkB were not changed when it was normalized to the level of TrkB expression. The difference on TrkB phosphorylation in Neuropep-1-injected rats may be affected by behavioral tests. These results suggest that Neuropep-1 may improve SLM via activation of the BDNF/TrkB signaling pathway in the rat hippocampus. Therefore, our findings represent that Neuropep-1 might be a potential candidate for treatment of learning and memory disorders as well as neurological diseases involving the abnormal expression of BDNF.