Figure 3 - uploaded by Noam E Ziv
Content may be subject to copyright.
Regional, cellular, and subcellular localization of Munc13-1–EYFP in Munc13-1– EYFP KI brains. A, Overview of direct Munc13-1–EYFP fluorescence in a sagittal section from a paraformaldehyde-fixed m/m mouse brain. Cer, Cerebellum; Co, cortex; Hi, hippocampus; OB, olfactory bulb; Sn, substantia nigra; St, striatum; Th, thalamus. Scale bar, 1 mm. B, C, Higherresolution images of direct Munc13-1–EYFP fluorescence in sections through the cerebellum of m/m (B) and WT (/) mice (C). g, Granule cell layer; m, molecular layer; p, Purkinje cell layer. Scale bars, 100 m. D, E, Higher-resolution images of direct Munc13-1–EYFP fluorescence in sections through the hippocampus of m/m (D) and WT (/) mice (E). hil, Hilus; sg, stratum granulosum; sl, stratum lucidum; sm, stratum moleculare; so, stratum oriens; sp, stratum pyramidale ; sr, stratum radiatum. Scale bars, 200 m. F, High-resolution image of direct Munc13- 1–EYFP fluorescence in CA3 region of the hippocampus of an m/m mouse. sl, Stratum lucidum; sp, stratum pyramidale. Scale bar, 25 m. G, Sagittal section through the brain of a WT mouse stained for Munc13-1 using HRP-coupled secondary antibodies and 3,3-diaminobenzidine (DAB). Note the similarity between this Munc13-1 immunostaining and direct Munc13-1–EYFP fluorescence (A). Scale bar, 1 mm. H, Images of sections through the CA3 region of the hippocampus of an m/m mouse after fluorescence immunostaining for the active zone marker Bassoon (red). Note the coincidence (yellow) of direct Munc13-1–EYFP fluorescence (green) and Bassoon-positive structures (red) in mossy fiber terminals of the stratum lucidum (sl). Scale bar, 10 m.
Source publication
GFP (green fluorescent protein) fusion proteins have revolutionized research on protein dynamics at synapses. However, corresponding analyses usually involve protein expression methods that override endogenous regulatory mechanisms, and therefore cause overexpression and temporal or spatial misexpression of exogenous fusion proteins, which may seri...
Contexts in source publication
Context 1
... cence sagittal brain sections from m/m mice that had been perfused with 4% para- formaldehyde. In all brain regions, the dis- tribution of Munc13-1-EYFP was identi- cal to that of typical general presynaptic marker proteins. Munc13-1-EYFP fluo- rescence was prominent in synaptic neu- ropil areas and absent from cell bodies and white matter (Fig. 3A). In the cerebellum, Munc13-1-EYFP was very abundant in the molecular layer, most likely represent- ing fluorescent parallel fiber terminals, less abundant in the granule cell layer, most likely reflecting labeled axon terminals in glomeruli, and absent from the Purkinje cell layer and white matter (Fig. 3B). Cor- responding sections ...
Context 2
... from cell bodies and white matter (Fig. 3A). In the cerebellum, Munc13-1-EYFP was very abundant in the molecular layer, most likely represent- ing fluorescent parallel fiber terminals, less abundant in the granule cell layer, most likely reflecting labeled axon terminals in glomeruli, and absent from the Purkinje cell layer and white matter (Fig. 3B). Cor- responding sections from WT cerebellum showed only background fluorescence (Fig. 3C). In the hippocampus, Munc13- 1-EYFP fluorescence was prominent in the stratum radiatum, stratum oriens, stratum lucidum, and hilus, which all con- tain dense synaptic neuropil (Fig. 3D). Lower level Munc13-1-EYFP fluorescence was detected in the ...
Context 3
... abundant in the molecular layer, most likely represent- ing fluorescent parallel fiber terminals, less abundant in the granule cell layer, most likely reflecting labeled axon terminals in glomeruli, and absent from the Purkinje cell layer and white matter (Fig. 3B). Cor- responding sections from WT cerebellum showed only background fluorescence (Fig. 3C). In the hippocampus, Munc13- 1-EYFP fluorescence was prominent in the stratum radiatum, stratum oriens, stratum lucidum, and hilus, which all con- tain dense synaptic neuropil (Fig. 3D). Lower level Munc13-1-EYFP fluorescence was detected in the stratum moleculare, and white matter was devoid of fluores- cence (Fig. 3D). Again, ...
Context 4
... in glomeruli, and absent from the Purkinje cell layer and white matter (Fig. 3B). Cor- responding sections from WT cerebellum showed only background fluorescence (Fig. 3C). In the hippocampus, Munc13- 1-EYFP fluorescence was prominent in the stratum radiatum, stratum oriens, stratum lucidum, and hilus, which all con- tain dense synaptic neuropil (Fig. 3D). Lower level Munc13-1-EYFP fluorescence was detected in the stratum moleculare, and white matter was devoid of fluores- cence (Fig. 3D). Again, sections from WT hippocampi only showed background flu- orescence (Fig. 3E). Overall, the distribution of Munc13-1-EYFP in brains from m/m mice was indistinguishable from the Munc13-1 ...
Context 5
... background fluorescence (Fig. 3C). In the hippocampus, Munc13- 1-EYFP fluorescence was prominent in the stratum radiatum, stratum oriens, stratum lucidum, and hilus, which all con- tain dense synaptic neuropil (Fig. 3D). Lower level Munc13-1-EYFP fluorescence was detected in the stratum moleculare, and white matter was devoid of fluores- cence (Fig. 3D). Again, sections from WT hippocampi only showed background flu- orescence (Fig. 3E). Overall, the distribution of Munc13-1-EYFP in brains from m/m mice was indistinguishable from the Munc13-1 distribution in WT mouse brains as detected by 3,3-diaminobenzidine im- munostaining with a Munc13-1-specific polyclonal antibody (Fig. ...
Context 6
... was prominent in the stratum radiatum, stratum oriens, stratum lucidum, and hilus, which all con- tain dense synaptic neuropil (Fig. 3D). Lower level Munc13-1-EYFP fluorescence was detected in the stratum moleculare, and white matter was devoid of fluores- cence (Fig. 3D). Again, sections from WT hippocampi only showed background flu- orescence (Fig. 3E). Overall, the distribution of Munc13-1-EYFP in brains from m/m mice was indistinguishable from the Munc13-1 distribution in WT mouse brains as detected by 3,3-diaminobenzidine im- munostaining with a Munc13-1-specific polyclonal antibody (Fig. ...
Context 7
... localization of Munc13-1- EYFP was most readily detectable in the stratum lucidum, where larger fluorescent puncta that most likely correspond to mossy fiber terminals were observed (Fig. 3F ). Colabeling of m/m brain sections with an antibody to the active zone marker Bassoon revealed an almost perfect colocal- ization of Munc13-1-EYFP with ...
Context 8
... characterization of Munc13-1-EYFP expression in whole- brain sections already indicated that the fusion protein is targeted properly to presynaptic terminals (Fig. 3H ). However, the reso- lution of this characterization is limited by the high density of synapses in sections. Therefore, we further characterized the cel- lular distribution of Munc13-1-EYFP in primary cultures of dis- sociated neurons prepared from the hippocampus and cerebral cortex of newborn Munc13-1-EYFP KI mice. As expected, ...
Context 9
... KI mice are viable, healthy, and show no defects in synaptic transmission. The fluorescence signals ob- tained from synapses in brain sections and cultured neurons are sufficiently high for detection by direct fluorescence (Fig. 3) and for imaging presynaptic Munc13-1-EYFP in living neurons (Figs. 5-7), although Munc13-1-EYFP expression levels do not exceed levels of WT Munc13-1. Although the fused EYFP cDNA appears to slightly affect Munc13-1 mRNA synthesis (Fig. 2 D), Munc13- 1-EYFP protein expression rates and steady-state expression lev- els in m/m mice are ...
Context 10
... of 3 min that represents 40% of total synaptic Munc13-1-EYFP, and a second pool, exchanged much more slowly (time constant of 80 min), that represents 60% of the total Munc13-1-EYFP (Fig. 7). We postulate that the quickly and slowly exchanging pools correspond to the biochemically distinguishable soluble and insoluble Munc13-1 pools, respectively (Fig. 3C). The quickly exchanging Munc13-1-EYFP pool most likely represents proteins whose diffusion is hindered by transient and weak pro- teinaceous interactions, because its exchange rate is still much slower than expected of free diffusion ( Tsuriel et al., 2006). In contrast, the slowly exchanging Munc13-1-EYFP pool may rep- resent ...
Similar publications
The master transcription factor SOX9 is a key player during chondrocyte differentiation, cartilage development, homeostasis and disease. Modulation of SOX9 and its target gene expression is essential during chondrogenic, osteogenic and adipogenic differentiation of human mesenchymal stem cells (hMSCs). However, lack of sufficient knowledge about th...
The combination of nanoscaled materials and biological self-assembly is a key step for the development of novel approaches for biotechnology and bionanoelectronic devices. Here we propose a route to merge these two subsystems and report on the formation of highly concentrated aqueous solutions of silanized silicon nanowires wrapped in a lipid bilay...
C. elegans has been an essential model organism in the fields of developmental biology, neuroscience, and aging. However, these areas have been limited by our ability to visualize and track individual C. elegans worms, especially at the subcellular scale, over the course of their lifetime. Here we present a microfluidic device to culture individual...
In this study we report new results on the changes in optical properties of glass surfaces induced during wet photolithography using Oblique Incidence Reflectivity Difference Ellipsometry (OI-RD). A novel wet UV-photolithographic method for patterning phospholipid bilayers into two-dimensional arrays of voids and patches on hydrophilic glass substr...
The motile characteristics and mechanisms that drive the dissemination of diffuse large B-cell lymphoma (DLBCL) are elusive. Here, we show that DLBCL initiates dissemination through activating STAT3-mediated amoeboid migration. Mechanistically, STAT3 activates RHOH transcription, which competes with the RhoGDP dissociation inhibitor RhoGDIγ to acti...
Citations
... Finally, it should be noted that next to the delivery of newly synthesised proteins from the soma, local shuffling of presynaptic material occurs, both between individual AZs, and between AZ and extrasynaptic, cytosolic reservoir pools [7,[43][44][45][46]. This local rearrangement of presynaptic material does not require an "on locus" delivery of a precise number of PVs to form an individual synapse. ...
The faithful formation and, consequently, function of a synapse requires continuous and tightly controlled delivery of synaptic material. At the presynapse, a variety of proteins with unequal molecular properties are indispensable to compose and control the molecular machinery concerting neurotransmitter release through synaptic vesicle fusion with the presynaptic membrane. As presynaptic proteins are produced mainly in the neuronal soma, they are obliged to traffic along microtubules through the axon to reach the consuming presynapse. This anterograde transport is performed by highly specialised and diverse presynaptic precursor vesicles, membranous organelles able to transport as different proteins such as synaptic vesicle membrane and membrane-associated proteins, cytosolic active zone proteins, ion-channels, and presynaptic membrane proteins, coordinating synaptic vesicle exo- and endocytosis. This review aims to summarise and categorise the diverse and numerous findings describing presynaptic precursor cargo, mode of trafficking, kinesin-based axonal transport and the molecular mechanisms of presynaptic precursor vesicles biogenesis in both vertebrate and invertebrate model systems.
... GFP-PATROL1 localizes on dot-like structures (GFP-PATROL1 dots) just beneath plasma membranes (Hashimoto-Sugimoto et al. 2013). These GFP-PATROL1 dots are stained by the endocytic tracer FM4-64 in leaf epidermal cells of A. thaliana (Hashimoto-Sugimoto et al. 2013), as is Munc13 in mouse brain cells (Kalla et al. 2006). Time-sequential imaging using a variable-angle epifluorescence microscope indicated that GFP-PATROL1 dots appear and remain at the same site for 2-10 s despite the existence of cytoplasmic streaming, suggesting that the dots are tethered to the plasma membranes and participate in membrane fusion (Higaki et al. 2014, Higaki 2015. ...
The Arabidopsis thaliana stomatal complex contains a pair of guard cells surrounded by subsidiary cells, which assist in turgor-driven stomatal movement and receive water and ions. This transport, driven by environmental signals, involves a translocation factor of the plasma membrane proton pump H⁺-ATPase AHA1, PATROL1. In this study, we investigated the responses of PATROL1 to salinity and hyperosmotic stresses. Specifically, we analyzed the effects of 125 mM NaCl or 231 mM mannitol on the cotyledon pavement cell cortexes in transgenic A. thaliana seedlings expressing green fluorescent protein (GFP)-tagged PATROL1. Cells treated with NaCl had few GFP-PATROL1-labeled dot-like structures but contained unusual labeled large bodies and rod-like structures. Cells treated with mannitol had similar large bodies, but not rods, indicating that the rod-like structures form specifically under salinity stress conditions. Dual observations of GFP-PATROL1 and red fluorescent protein (RFP)-tagged AHA1 in stress-treated cells revealed that the latter did not accumulate in the stress-induced GFP-PATROL1 structures, suggesting that the stress-induced GFP-PATROL1 structures are not involved in RFP-AHA1 localization. Additionally, the primary root growth of the patrol1 mutant was more sensitive to NaCl treatment than was that of wild type. Thus, PATROL1 appears to contribute to salinity stress tolerance, possibly by regulating membrane trafficking.
... Finally, a robust retrograde transport of AZ protein suggest that AZ proteins marked for degradation are most likely targeted retrogradely to the cell soma for destruction via the proteasome pathway. In fact, AZ may be a highly dynamic structure, with proteins getting exchanged between synapses in central neurons [41]. In NMJ, where synapses are at the terminal far from the cell soma, such local exchanges may be possible but the retrograde trafficking is intriguing and may indicate a low proteasomal activity at NMJ presynaptic compartment. ...
Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles (SV) and released at the synaptic active zone (AZ). While in recent years major progress has been made in unraveling the molecular machinery responsible for SV docking, fusion and exocytosis, the mechanisms governing AZ protein and SV trafficking through axons still remain unclear. Here, we performed stop-flow nerve ligation to examine axonal trafficking of endogenous AZ and SV proteins. Rat sciatic nerves were collected 1 h, 3 h and 8 h post ligation and processed for immunohistochemistry and electron microscopy. First, we followed the transport of an integral synaptic vesicle protein, SV2A and a SV-associated protein involved in SV trafficking, Rab3a, and observed that while SV2A accumulated on both sides of ligation, Rab3a was only noticeable in the proximal segment of the ligated nerve indicating that only SV trans-membrane protein SV2A displayed a bi-directional axonal transport. We then demonstrate that multiple AZ proteins accumulate rapidly on either side of the ligation with a timescale similar to that of SV2A. Overall, our data uncovers an unexpected robust bi-directional, coordinated -trafficking of SV and AZ proteins in peripheral nerves. This implies that pathological disruption of axonal trafficking will not only impair trafficking of newly synthesized proteins to the synapse but will also affect retrograde transport, leading to neuronal dysfunction and likely neurodegeneration.
... FRAP showed that SYD-2 and ELKS-1 were dynamic at these nascent synapses (Fig. 1a, b), with recovery times comparable to those of other cellular LLPSs [11][12][13] . By contrast, SYD-2 and ELKS-1 were extremely static at mature nerve-ring synapses Article ( Fig. 1a, b), consistent with the slow dynamics of active-zone proteins seen at synapses established by cultured vertebrate neurons 14,15 . Furthermore, in the outgrowing PDE neuron 4 , SYD-2 and ELKS-1 were more dynamic at nascent synapses near the growth cone than at older synapses along the same axon close to the cell body (Fig. 1c, d). ...
The formation of synapses during neuronal development is essential for establishing neural circuits and a nervous system¹. Every presynapse builds a core ‘active zone’ structure, where ion channels cluster and synaptic vesicles release their neurotransmitters². Although the composition of active zones is well characterized2,3, it is unclear how active-zone proteins assemble together and recruit the machinery required for vesicle release during development. Here we find that the core active-zone scaffold proteins SYD-2 (also known as liprin-α) and ELKS-1 undergo phase separation during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation by using mutant SYD-2 and ELKS-1 proteins that specifically lack this activity. These mutant proteins remain enriched at synapses in Caenorhabditis elegans, but show defects in active-zone assembly and synapse function. The defects are rescued by introducing a phase-separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid-phase scaffold, and find that it is competent to bind and incorporate downstream active-zone components. We find that the fluidity of SYD-2 and ELKS-1 condensates is essential for efficient mixing and incorporation of active-zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for the assembly of the synaptic active zone, before maturation into a stable final structure.
... High expression levels of the tagged proteins are beneficial for their visualization, but can also disturb the biochemical homeostasis of the target cells by interfering with the endogenous gene regulatory mechanisms. This may result in artificial conditions, which are difficult to interpret [2]. To study fluorescently tagged proteins in their native environment under the control of their endogenous regulatory elements, the generation of knock-in (KI) mice in which target proteins are manipulated in their endogenous loci is better suited. ...
... This strategy was successfully employed to generate KI mice for the analysis of Munc13 isoforms [2]. In vertebrates, four Munc13 isoforms (Munc13-1, -2, -3, and -4) are known. ...
... For each of the three neuronally expressed Munc13 isoforms, KI mouse lines were generated by inserting a sequence coding for GFP or YFP at their respective genomic loci. This resulted in the expression of C-terminally tagged fluorescent Munc13-EXFP fusion proteins at endogenous concentrations and locations, i.e., Munc13-1-EYFP, Munc13-2-EYFP and Munc13-3-EGFP, which still underlie all cell type-specific regulatory mechanisms [2,14]. For the Munc13-1-EYFP mouse line, it was shown that the endogenous expression level of the tagged Munc13-1 isoform was sufficient for direct visualization of active zones at brain synapses, and electrophysiological measurements suggested that neurons and synapses in Munc13-1-EYFP mice are capable of normal synaptic transmission [2]. ...
Munc13 isoforms are constituents of the presynaptic compartment of chemical synapses, where they govern important steps in preparing synaptic vesicles for exocytosis. The role of Munc13-1,-2 and-3 is well documented in brain neurons, but less is known about their function and distribution among the neurons of the retina and their conventional and ribbon-type chemical synapses. Here, we examined the retinae of Munc13-1- ,-2-, and-3-EXFP knock-in (KI) mice with a combination of immunocytochemistry, physiology, and electron microscopy. We show that knock-in of Munc13-EXFP fusion proteins did not affect overall retinal anatomy or synapse structure, but slightly affected synaptic transmission. By labeling Munc13-EXFP KI retinae with specific antibodies against Munc13-1,-2 and-3, we found that unlike in the brain, most retinal synapses seem to operate with a single Munc13 isoform. A surprising exception to this rule was type 6 ON bipolar cells, which expressed two Munc13 isoforms in their synaptic terminals, ubMunc13-2 and Munc13-3. The results of this study provide an important basis for future studies on the contribution of Munc13 isoforms in visual signal processing in the mammalian retina.
... Several such studies were reproduced well by our data (Appendix Fig S6). At the same time, a FRAP measurement of knock-in Munc13 provided a substantially lower mobility (Kalla et al, 2006), with the shortest time constant measured in cultured mouse cortical neurons being around~3 min, as opposed to a few seconds in our measurements. This difference probably has both technical and biological grounds. ...
Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology.
... Other molecules, however, might be affected differently. For example, prolonged silencing was shown to slow the exchange kinetics of Shank3/ProSAP2 and Munc13-1 (Kalla et al., 2006;Tsuriel et al., 2006); thus, relationships between molecular dynamics and size distributions might differ among synaptic molecules. Finally, many additional mechanisms have been implicated in synaptic size/strength distribution scaling (Turrigiano, 2008;Pozo and Goda, 2010;Chowdhury and Hell, 2018) including mechanisms directly involving PSD-95 (Sun and Turrigiano, 2011; further highlighting the explanatory challenges these phenomena pose. ...
The extraordinary diversity of excitatory synapse sizes is commonly attributed to activity-dependent processes that drive synaptic growth and diminution. Recent studies also point to activity-independent size fluctuations, possibly driven by innate synaptic molecule dynamics, as important generators of size diversity. To examine the contributions of activity-dependent and independent processes to excitatory synapse size diversity, we studied glutamatergic synapse size dynamics and diversification in cultured rat cortical neurons (both sexes), silenced from plating. We found that in networks with no history of activity whatsoever, synaptic size diversity was no less extensive than that observed in spontaneously active networks. Synapses in silenced networks were larger, size distributions were broader, yet these were rightward-skewed and similar in shape when scaled by mean synaptic size. Silencing reduced the magnitude of size fluctuations and weakened constraints on size distributions, yet these were sufficient to explain synaptic size diversity in silenced networks. Model-based exploration followed by experimental testing indicated that silencing-associated changes in innate molecular dynamics and fluctuation characteristics might negatively impact synaptic persistence, resulting in reduced synaptic numbers. This, in turn, would increase synaptic molecule availability, promote synaptic enlargement, and ultimately alter fluctuation characteristics. These findings suggest that activity-independent size fluctuations are sufficient to fully diversify glutamatergic synaptic sizes, with activity-dependent processes primarily setting the scale rather than the shape of size distributions. Moreover, they point to reciprocal relationships between synaptic size fluctuations, size distributions, and synaptic numbers mediated by the innate dynamics of synaptic molecules as they move in, out, and between synapses.
... The next day, the SC WT cells were added to the DRG neurons and co-cultured for another six to seven days, allowing functional synapses to form. On DIV6 or 7, cells were stained with antibodies against CAPS1 (Farina et al., 2015; and Munc13-1 (Farina et al., 2015), which is a well described priming protein for SV and LDCV exocytosis in neurons (Varoqueaux et al., 2002;van de Bospoort et al., 2012) and is specifically localized to active zones (Kalla et al., 2006). ...
... Concerning the target-selectivity and the timing of NGFdependent, UPS-mediated presynaptic degradation at cholinergic nerve endings, it's worth noticing that -although UPS activity is localized in synaptic terminals (Chain et al., 1995;Patrick, 2006) and synaptic activity promotes UPS sequestration within dendritic spines (Bingol and Schuman, 2006)-only few synaptic proteins actually undergo local, activity-regulated and UPSmediated degradation (Colledge et al., 2003;Ehlers, 2003;Bingol and Schuman, 2005;Jiang et al., 2010;Shin et al., 2012;Alvarez-Castelao and Schuman, 2015). Interestingly, under basal conditions, both pharmacological and genetic suppression of proteasomal activity does not significantly change the constitutive degradation rates in the bulk of resident synaptic proteins up to 10-24 h (Kalla et al., 2006;Shin et al., 2012;Hakim et al., 2016). Consistently, the local, activity-inducible, UPS-mediated degradation at terminal ends appears to be devoted mainly to reshape the synaptic structure/functions within a well-defined and narrow spatio-temporal window (Hanus and Schuman, 2013), while the constitutive clearance of the majority of synaptically residing proteins occurs along different intracellular degradative pathways (Cohen and Ziv, 2017). ...
Basal forebrain cholinergic neurons (BFCNs) depend on nerve growth factor (NGF) for their survival/differentiation and innervate cortical and hippocampal regions involved in memory/learning processes. Cholinergic hypofunction and/or degeneration early occurs at prodromal stages of Alzheimer’s disease (AD) neuropathology in correlation with synaptic damages, cognitive decline and behavioral disability. Alteration(s) in ubiquitin-proteasome system (UPS) is also a pivotal AD hallmark but whether it plays a causative, or only a secondary role, in early synaptic failure associated with disease onset remains unclear. We previously reported that impairment of NGF/TrkA signaling pathway in cholinergic-enriched septo-hippocampal primary neurons triggers “dying-back” degenerative processes which occur prior to cell death in concomitance with loss of specific vesicle trafficking proteins, including synapsin I, SNAP-25 and α-synuclein, and with deficit in presynaptic excitatory neurotransmission. Here, we show that in this in vitro neuronal model: (i) UPS stimulation early occurs following neurotrophin starvation (-1 h up to -6 h); (ii) NGF controls the steady-state levels of these three presynaptic proteins by acting on coordinate mechanism(s) of dynamic ubiquitin-C-terminal hydrolase 1 (UCHL-1)-dependent (mono)ubiquitin turnover and UPS-mediated protein degradation. Importantly, changes in miniature excitatory post-synaptic currents (mEPSCs) frequency detected in -6 h NGF-deprived primary neurons are strongly reverted by acute inhibition of UPS and UCHL-1, indicating that NGF tightly controls in vitro the presynaptic efficacy via ubiquitination-mediated pathway(s). Finally, changes in synaptic ubiquitin and selective reduction of presynaptic markers are also found in vivo in cholinergic nerve terminals from hippocampi of transgenic Tg2576 AD mice, even from presymptomatic stages of neuropathology (1-month-old). By demonstrating a crucial role of UPS in the dysregulation of NGF/TrkA signaling on properties of cholinergic synapses, these findings from two well-established cellular and animal AD models provide novel therapeutic targets to contrast early cognitive and synaptic dysfunction associated to selective degeneration of BFCNs occurring in incipient early/middle-stage of disease.
... In mature hippocampal synapses, liprin-α2 was found to be a very dynamic protein in comparison with Munc13 and Bassoon, which are very stable [91,92], and through its interactions with RIM1 and CASK, it regulates presynaptic organization and hence SV release in response to network activity [93,94]. Elimination of liprin-α2 by knockdown in mature hippocampal neurons does not affect the number of active synapses but does alter the efficiency of SV release by regulating RRP size. ...
Among all the biological systems in vertebrates, the central nervous system (CNS) is the most complex, and its function depends on specialized contacts among neurons called synapses. The assembly and organization of synapses must be exquisitely regulated for a normal brain function and network activity. There has been a tremendous effort in recent decades to understand the molecular and cellular mechanisms participating in the formation of new synapses and their organization, maintenance, and regulation. At the vertebrate presynapses, proteins such as Piccolo, Bassoon, RIM, RIM-BPs, CAST/ELKS, liprin-α, and Munc13 are constant residents and participate in multiple and dynamic interactions with other regulatory proteins, which define network activity and normal brain function. Here, we review the function of these active zone (AZ) proteins and diverse factors involved in AZ assembly and maintenance, with an emphasis on axonal trafficking of precursor vesicles, protein homo- and hetero-oligomeric interactions as a mechanism of AZ trapping and stabilization, and the role of F-actin in presynaptic assembly and its modulation by Wnt signaling.
