Project description:The CREB-binding protein (CBP) exerts tight control of developmental processes. Here, we investigated the consequences of its selective ablation in newborn neurons. Mice in which CBP was eliminated during neuronal differentiation showed perinatal death and defective diaphragm innervation. Adult-born neurons also showed impaired growth and maturation after inducible and restricted CBP loss in dentate gyrus neuroprogenitors. Consistent with these in vivo findings, cultured neurons displayed impaired outgrowth, immature spines, and deficient activity-dependent synaptic remodeling after CBP ablation. These deficits coincided with broad transcriptional changes affecting genes involved in neuronal growth and plasticity. The affected gene set included many predicted targets of both CBP and the serum response factor (SRF), an activity-regulated transcription factor involved in structural plasticity. Notably, increasing SRF activity in a CBP-independent manner ameliorated the transcriptional, synaptic, and growth defects. These results underscore the relevance of CBP-SRF interactions during neuronal outgrowth and synaptic maturation, and demonstrate that CBP plays an essential role in supporting the gene program underlying the last steps of neuronal differentiation, both during development and in the adult brain.
Project description:Regulation of synaptic morphology depends on endocytosis of activated growth signal receptors, but the mechanisms regulating this membrane-trafficking event are unclear. Actin polymerization mediated by Wiskott-Aldrich syndrome protein (WASp) and the actin-related protein 2/3 complex generates forces at multiple stages of endocytosis. FCH-BIN amphiphysin RVS (F-BAR)/SH3 domain proteins play key roles in this process by coordinating membrane deformation with WASp-dependent actin polymerization. However, it is not known how other WASp ligands, such as the small GTPase Cdc42, coordinate with F-BAR/SH3 proteins to regulate actin polymerization at membranes. Nervous Wreck (Nwk) is a conserved neuronal F-BAR/SH3 protein that localizes to periactive zones at the Drosophila larval neuromuscular junction (NMJ) and is required for regulation of synaptic growth via bone morphogenic protein signaling. Here, we show that Nwk interacts with the endocytic proteins dynamin and Dap160 and functions together with Cdc42 to promote WASp-mediated actin polymerization in vitro and to regulate synaptic growth in vivo. Cdc42 function is associated with Rab11-dependent recycling endosomes, and we show that Rab11 colocalizes with Nwk at the NMJ. Together, our results suggest that synaptic growth activated by growth factor signaling is controlled at an endosomal compartment via coordinated Nwk and Cdc42-dependent actin assembly.
Project description:Mutations in genes essential for protein homeostasis have been identified in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) patients. Why mature neurons should be particularly sensitive to such perturbations is unclear. We identified mutations in Rab8 in a genetic screen for enhancement of an FTD phenotype associated with ESCRT-III dysfunction. Examination of Rab8 mutants or motor neurons expressing a mutant ESCRT-III subunit, CHMP2B(Intron5), at the Drosophila melanogaster neuromuscular junction synapse revealed synaptic overgrowth and endosomal dysfunction. Expression of Rab8 rescued overgrowth phenotypes generated by CHMP2B(Intron5). In Rab8 mutant synapses, c-Jun N-terminal kinase (JNK)/activator protein-1 and TGF-β signaling were overactivated and acted synergistically to potentiate synaptic growth. We identify novel roles for endosomal JNK-scaffold POSH (Plenty-of-SH3s) and a JNK kinase kinase, TAK1, in regulating growth activation in Rab8 mutants. Our data uncover Rab8, POSH, and TAK1 as regulators of synaptic growth responses and point to recycling endosome as a key compartment for synaptic growth regulation during neurodegenerative processes.
Project description:BackgroundThe growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance.ResultsWe identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants - there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth.ConclusionThe F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses.
Project description:Local translation of mRNAs in the synapse has a major role in synaptic structure and function. Chronic alcohol use causes persistent changes in synaptic mRNA expression, possibly mediated by microRNAs localized in the synapse. We profiled the transcriptome of synaptoneurosomes (SN) obtained from the amygdala of mice that consumed 20% ethanol (alcohol) in a 30-day continuous two-bottle choice test to identify the microRNAs that target alcohol-induced mRNAs. SN are membrane vesicles containing pre- and post-synaptic compartments of neurons and astroglia and are a unique model for studying the synaptic transcriptome. We previously showed that chronic alcohol regulates mRNA expression in a coordinated manner. Here, we examine microRNAs and mRNAs from the same samples to define alcohol-responsive synaptic microRNAs and their predicted interactions with targeted mRNAs. The aim of the study was to identify the microRNA-mRNA synaptic interactions that are altered by alcohol. This was accomplished by comparing the effect of alcohol in SN and total homogenate preparations from the same samples. We used a combination of unbiased bioinformatic methods (differential expression, correlation, co-expression, microRNA-mRNA target prediction, co-targeting, and cell type-specific analyses) to identify key alcohol-sensitive microRNAs. Prediction analysis showed that a subset of alcohol-responsive microRNAs was predicted to target many alcohol-responsive mRNAs, providing a bidirectional analysis for identifying microRNA-mRNA interactions. We found microRNAs and mRNAs with overlapping patterns of expression that correlated with alcohol consumption. Cell type-specific analysis revealed that a significant number of alcohol-responsive mRNAs and microRNAs were unique to glutamate neurons and were predicted to target each other. Chronic alcohol consumption appears to perturb the coordinated microRNA regulation of mRNAs in SN, a mechanism that may explain the aberrations in synaptic plasticity affecting the alcoholic brain.
Project description:The death-associated protein kinase 1 (DAPK1) regulates the synaptic movement of the Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII). Synaptic CaMKII accumulation is mediated via binding to the NMDA-receptor subunit GluN2B and is required for long-term potentiation (LTP). By contrast, long-term depression (LTD) instead requires specific suppression of this movement, which is mediated by competitive DAPK1 binding to GluN2B. We find here that DAPK1 localizes to synapses via two distinct mechanisms: basal localization requires F-actin, but retention of DAPK1 at synapses during LTD requires an additional binding mode, likely to GluN2B. While F-actin binding mediates DAPK1 enrichment at synapses, it is not sufficient to suppress synaptic CaMKII movement. However, it is a prerequisite that enables the additional LTD-specific binding mode of DAPK1, which in turn mediates suppression of the CaMKII movement. Thus, both modes of synaptic DAPK1 localization work together to regulate synaptic CaMKII localization and thereby synaptic plasticity.
Project description:PSD-95 MAGUK family scaffold proteins are multi-domain organisers of synaptic transmission that contain three PDZ domains followed by an SH3-GK domain tandem. This domain architecture allows coordinated assembly of protein complexes composed of neurotransmitter receptors, synaptic adhesion molecules and downstream signalling effectors. Here we show that binding of monomeric CRIPT-derived PDZ3 ligands to the third PDZ domain of PSD-95 induces functional changes in the intramolecular SH3-GK domain assembly that influence subsequent homotypic and heterotypic complex formation. We identify PSD-95 interactors that differentially bind to the SH3-GK domain tandem depending on its conformational state. Among these interactors, we further establish the heterotrimeric G protein subunit Gnb5 as a PSD-95 complex partner at dendritic spines of rat hippocampal neurons. The PSD-95 GK domain binds to Gnb5, and this interaction is triggered by CRIPT-derived PDZ3 ligands binding to the third PDZ domain of PSD-95, unraveling a hierarchical binding mechanism of PSD-95 complex formation.
Project description:SNAP-25 exists as two developmentally regulated alternatively spliced isoforms, SNAP-25a and SNAP-25b. We explored the function of SNAP-25a and SNAP-25b at Schaffer collateral-CA1 synapses in hippocampus using 4-week-old wild-type (WT) and SNAP-25b-deficient (MT) mice. Characterizing the protein expression of individual SNAP-25 isoforms revealed that WT females had higher levels of SNAP-25a than WT males, suggesting a sex-dependent delay of the alternative splicing switch from SNAP-25a to SNAP-25b. MT mice expressed normal levels of total SNAP-25, Syntaxin 1A and SNAP-47 in the hippocampus, but females expressed lower levels of VAMP2. Electrophysiological recordings in in vitro hippocampal slices revealed significantly reduced magnitude of LTP in MT mice. We also found reduction in paired-pulse facilitation after induction of LTP in WT males, but not in WT females, possibly related to the difference in SNAP-25a/SNAP-25b ratios, suggesting that the splicing switch may play a sex-specific role in LTP-associated increases in presynaptic release probability. Basal synaptic transmission measured in input-output relations revealed that the ability to discriminate between the intensity of presynaptic stimuli was affected in SNAP-25b-deficient mice. Learning in a behavioural paradigm of active-avoidance was impaired in MT mice, strengthening the conclusion that SNAP-25b is important for cognitive performance by altering activity-dependent synaptic plasticity.
Project description:Sensory hair cells in the ear utilize specialized ribbon synapses. These synapses are defined by electron-dense presynaptic structures called ribbons, composed primarily of the structural protein Ribeye. Previous work has shown that voltage-gated influx of Ca2+ through CaV1.3 channels is critical for hair-cell synapse function and can impede ribbon formation. We show that in mature zebrafish hair cells, evoked presynaptic-Ca2+ influx through CaV1.3 channels initiates mitochondrial-Ca2+ (mito-Ca2+) uptake adjacent to ribbons. Block of mito-Ca2+ uptake in mature cells depresses presynaptic-Ca2+ influx and impacts synapse integrity. In developing zebrafish hair cells, mito-Ca2+ uptake coincides with spontaneous rises in presynaptic-Ca2+ influx. Spontaneous mito-Ca2+ loading lowers cellular NAD+/NADH redox and downregulates ribbon size. Direct application of NAD+ or NADH increases or decreases ribbon size respectively, possibly acting through the NAD(H)-binding domain on Ribeye. Our results present a mechanism where presynaptic- and mito-Ca2+ couple to confer proper presynaptic function and formation.
Project description:ObjectiveHomeostatic synaptic plasticity (HSP) serves as a gain control mechanism at central nervous system (CNS) synapses, including those between the dentate gyrus (DG) and CA3. Improper circuit control of DG-CA3 synapses is hypothesized to underlie epileptogenesis. Here, we sought to (1) identify compounds that preferentially modulate DG-CA3 synapses in primary neuronal culture and (2) determine if these compounds would delay or prevent epileptogenesis in vivo.MethodsWe previously developed and validated an in vitro assay to visualize the behavior of DG-CA3 synapses and predict functional changes. We used this "synapse-on-chip" assay (quantification of synapse size, number, and type using immunocytochemical markers) to dissect the mechanisms of HSP at DG-CA3 synapses. Using chemogenetic constructs and pharmacological agents we determined the signaling cascades necessary for gain control at DG-CA3 synapses. Finally, we tested the implicated cascades (using kappa opioid receptor (OR) agonists and antagonists) in two models of epileptogenesis: electrical amygdala kindling in the mouse and chemical (pentylenetetrazole) kindling in the rat.ResultsIn vitro, synapses between DG mossy fibers (MFs) and CA3 neurons are the primary homeostatic responders during sustained periods of activity change. Kappa OR signaling is both necessary and sufficient for the homeostatic elaboration of DG-CA3 synapses, induced by presynaptic DG activity levels. Blocking kappa OR signaling in vivo attenuates the development of seizures in both mouse and rat models of epilepsy.SignificanceThis study elucidates mechanisms by which synapses between DG granule cells and CA3 pyramidal neurons undergo activity-dependent homeostatic compensation, via OR signaling in vitro. Modulation of kappa OR signaling in vivo alters seizure progression, suggesting that breakdown of homeostatic closed-loop control at DG-CA3 synapses contributes to seizures, and that targeting endogenous homeostatic mechanisms at DG-CA3 synapses may prove useful in combating epileptogenesis.