The extracellular matrix (ECM) is a complex molecular network distributed throughout the extracellular space of different tissues as well as the neuronal system. Previous studies have identified ...various ECM components that play important roles in neuronal maturation and signal transduction. ECM components are reported to be involved in neurogenesis, neuronal migration, and axonal growth by interacting or binding to specific receptors. In addition, the ECM is found to regulate synapse formation, the stability of the synaptic structure, and synaptic plasticity. Here, we mainly reviewed the effects of various ECM components on synapse formation and briefly described the related diseases caused by the abnormality of several ECM components.
Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ...ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2×10(8) TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ≈1.3 days) or subjected to multiple freeze-thaw cycles (τ=1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.
Three neuronal pentraxins are expressed in brain, the membrane-bound "neuronal pentraxin receptor" (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at ...excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1β-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses.
Neuronal pentraxins comprise three neuronal proteins, neuronal pentraxin receptor (NPR) which is a type-II transmembrane protein on the neuronal surface, and secreted neuronal pentraxin-1 and NARP. The general functions of neuronal pentraxins at synapses have not been explored, except for their basic AMPAR binding properties. Here, we examined the functional role of NPR at synapses because it is the only neuronal pentraxin that is anchored to the neuronal cell-surface membrane. We find that NPR is a potent inducer of both excitatory and inhibitory heterologous synapses, and that knockdown of NPR in cultured neurons decreases the density of both excitatory and inhibitory synapses. Our data suggest that NPR performs a general, previously unrecognized function as a universal organizer of synapses.
Synapse is the fundamental structure for neurons to transmit information between cells. The proper synapse formation is crucial for developing neural circuits and cognitive functions of the brain. ...The aberrant synapse formation has been proved to cause many neurological disorders, including autism spectrum disorders and intellectual disability. Synaptic cell adhesion molecules (CAMs) are thought to play a major role in achieving mechanistic cell-cell recognition and initiating synapse formation via trans-synaptic interactions. Due to the diversity of synapses in different brain areas, circuits and neurons, although many synaptic CAMs, such as Neurexins (NRXNs), Neuroligins (NLGNs), Synaptic cell adhesion molecules (SynCAMs), Leucine-rich-repeat transmembrane neuronal proteins (LRRTMs), and SLIT and NTRK-like protein (SLITRKs) have been identified as synaptogenic molecules, how these molecules determine specific synapse formation and whether other molecules driving synapse formation remain undiscovered are unclear. Here, to provide a tool for synapse labeling and synaptic CAMs screening by artificial synapse formation (ASF) assay, we generated synaptotagmin-1-tdTomato (
tdTomato) transgenic mice by inserting the tdTomato-fused synaptotagmin-1 coding sequence into the genome of C57BL/6J mice. In the brain of
tdTomato transgenic mice, the tdTomato-fused synaptotagmin-1 (SYT1-tdTomato) signals were widely observed in different areas and overlapped with synapsin-1, a widely-used synaptic marker. In the olfactory bulb, the SYT1-tdTomato signals are highly enriched in the glomerulus. In the cultured hippocampal neurons, the SYT1-tdTomato signals showed colocalization with several synaptic markers. Compared to the wild-type (WT) mouse neurons, cultured hippocampal neurons from
tdTomato transgenic mice presented normal synaptic neurotransmission. In ASF assays, neurons from
tdTomato transgenic mice could form synaptic connections with HEK293T cells expressing NLGN2, LRRTM2, and SLITRK2 without immunostaining. Therefore, our work suggested that the
tdTomato transgenic mice with the ability to label synapses by tdTomato, and it will be a convenient tool for screening synaptogenic molecules.
N-methyl-D-aspartate receptors (NMDARs) are essential for excitatory neurotransmission and synaptic plasticity. GluN2A and GluN2B, two predominant Glu2N subunits of NMDARs in the hippocampus and the ...cortex, display distinct clustered distribution patterns and mobility at synaptic and extrasynaptic sites. However, how GluN2A clusters are specifically organized and stabilized remains poorly understood. Here, we found that the previously reported GluN2A-specific binding partner Rabphilin-3A (Rph3A) has the ability to undergo phase separation, which relies on arginine residues in its N-terminal domain. Rph3A phase separation promotes GluN2A clustering by binding GluN2A's C-terminal domain. A complex formed by Rph3A, GluN2A, and the scaffolding protein PSD95 promoted Rph3A phase separation. Disrupting Rph3A's phase separation suppressed the synaptic and extrasynaptic surface clustering, synaptic localization, stability, and synaptic response of GluN2A in hippocampal neurons. Together, our results reveal the critical role of Rph3A phase separation in determining the organization and stability of GluN2A in the neuronal surface.
Recent genome-wide association studies (GWAS) have identified multiple risk loci that show strong associations with schizophrenia. However, pinpointing the potential causal genes at the reported loci ...remains a major challenge. Here we identify candidate causal genes for schizophrenia using an integrative genomic approach. Sherlock integrative analysis shows that ALMS1, GLT8D1, and CSNK2B are schizophrenia risk genes, which are validated using independent brain expression quantitative trait loci (eQTL) data and integrative analysis method (SMR). Consistently, gene expression analysis in schizophrenia cases and controls further supports the potential role of these three genes in the pathogenesis of schizophrenia. Finally, we show that GLT8D1 and CSNK2B knockdown promote the proliferation and inhibit the differentiation abilities of neural stem cells, and alter morphology and synaptic transmission of neurons. These convergent lines of evidence suggest that the ALMS1, CSNK2B, and GLT8D1 genes may be involved in pathophysiology of schizophrenia.
Efficient optical clearance is fundamental for whole brain imaging. In particular, clearance of the brain without membrane damage is required for the imaging of lipophilic tracer-labeled neural ...tracts. Relying on an ascending gradient of fructose solutions, SeeDB can achieve sufficient transparency of the mouse brain while ensuring that the plasma membrane remains intact. However, it is challenging to extend this method to larger mammalian brains due to the extremely high viscosity of the saturated fructose solution. Here we report a SeeDB-derived optical clearing method, termed FRUIT, which utilizes a cocktail of fructose and urea. As demonstrated in the adult mouse brain, combination of these two highly water-soluble clearing agents exerts a synergistic effect on clearance. More importantly, the final FRUIT solution has low viscosity so as to produce transparency of the whole adult rabbit brain via arterial perfusion, which is impossible to achieve with a saturated fructose solution. In addition to good compatibility with enhanced yellow fluorescent protein, the cocktail also preserves the fluorescence of the lipophilic tracer DiI. This work provides a volume-independent optical clearing method which retains the advantages of SeeDB, particularly compatibility with lipophilic tracers.
Dendritic spines are actin-rich membrane protrusions that are the major sites of excitatory synaptic input in the mammalian brain, and their morphological plasticity provides structural basis for ...learning and memory. Here we report that endophilin A1, with a well-established role in clathrin-mediated synaptic vesicle endocytosis at the pre- synaptic terminal, also localizes to dendritic spines and is required for spine morphogenesis, synapse formation and synaptic function. We identify pl40Cap, a regulator of cytoskeleton reorganization, as a downstream effector of en- dophilin A1 and demonstrate that disruption of their interaction impairs spine formation and maturation. Moreover, we demonstrate that knockdown of endophUin A1 or pl40Cap impairs spine stabilization and synaptic function. We further show that endophilin A1 regulates the distribution of pl40Cap and its downstream effector, the F-actin-binding protein cortactin as well as F-actin enrichment in dendritic spines. Together, these results reveal a novel function of postsynaptic endophilin A1 in spine morphogenesis, stabilization and synaptic function through the regulation of pl40Cap.
Purkinje cells (PCs) in the cerebellum receive two excitatory afferents including granule cells-derived parallel fiber (PF) and the climbing fiber. Scaffolding protein Rack1 is highly expressed in ...the cerebellar PCs. Here, we found delayed formation of specific cerebellar vermis lobule and impaired motor coordination in PC-specific Rack1 conditional knockout mice. Our studies further revealed that Rack1 is essential for PF-PC synapse formation. In addition, Rack1 plays a critical role in regulating synaptic plasticity and long-term depression (LTD) induction of PF-PC synapses without changing the expression of postsynaptic proteins. Together, we have discovered Rack1 as the crucial molecule that controls PF-PC synaptogenesis and synaptic plasticity. Our studies provide a novel molecular insight into the mechanisms underlying the neural development and neuroplasticity in the cerebellum.
In the original version of this Article, the affiliation details for Qiushuo Shen incorrectly omitted 'Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, 650204, ...China'. This has now been corrected in both the PDF and HTML versions of the Article.
PDF
