Noam E. Ziv

Assembly of New Individual Excitatory Synapses: Time Course and Temporal Order of Synaptic Molecule Recruitment

Time-lapse microscopy, retrospective immunohistochemistry, and cultured hippocampal neurons were used to determine the time frame of individual glutamatergic synapse assembly and the temporal order in which specific molecules accumulate at new synaptic junctions. New presynaptic boutons capable of activity-evoked vesicle recycling were observed to form within 30 min of initial axodendritic contact. Clusters of the presynaptic active zone protein Bassoon were present in all new boutons. Conversely, clusters of the postsynaptic molecule SAP90/PSD-95 and glutamate receptors were found on average only approximately 45 min after such boutons were first detected. AMPA- and NMDA-type glutamate receptors displayed similar clustering kinetics. These findings suggest that glutamatergic synapse assembly can occur within 1-2 hr after initial contact and that presynaptic differentiation may precede postsynaptic differentiation.

Assembling the Presynaptic Active Zone: A Characterization of an Active Zone Precursor Vesicle

The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.

Unitary Assembly of Presynaptic Active Zones from Piccolo-Bassoon Transport Vesicles

Recent studies indicate that active zones (AZs)-sites of neurotransmitter release-may be assembled from preassembled AZ precursor vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor vesicle of CNS synapses-the Piccolo-Bassoon transport vesicle (PTV)-carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.

Cellular and molecular mechanisms of presynaptic assembly

The formation of a presynaptic neurotransmitter secretion site involves the transformation of a patch of unspecialized plasma membrane into a complex structure that is highly specialized for sustained, depolarization-evoked synaptic vesicle exocytosis. This transformation is governed by dynamic intracellular and intercellular processes, and by global developmental processes. Recent work has shed light on the cellular processes that are responsible for transporting presynaptic molecules to nascent presynaptic sites and for the subsequent transformation of these sites into functional presynaptic structures. Other studies are providing clues to the identity of molecules that induce and initiate the transformation process.

Molecular mechanisms of CNS synaptogenesis

Synapses of the mammalian CNS are asymmetric sites of cell-cell adhesion between nerve cells. They are designed to mediate the rapid and efficient transmission of signals from the presynaptic bouton of one neuron to the postsynaptic plasma membrane of a second neuron. Significant progress has been made in the characterization of the structural, functional and developmental assembly of CNS synapses. Recent progress has been made in understanding the molecular and cellular mechanisms that underlie synaptogenesis, in particular that of glutamatergic synapses of the CNS.

Postsynaptic Density Assembly Is Fundamentally Different from Presynaptic Active Zone Assembly

The cellular mechanisms involved in the formation of the glutamatergic postsynaptic density (PSD) are mainly unknown. Previous studies have indicated that PSD assembly may occur in situ by a gradual recruitment of postsynaptic molecules, whereas others have suggested that the PSD may be assembled from modular transport packets assembled elsewhere. Here we used cultured hippocampal neurons and live cell imaging to examine the process by which PSD molecules from different layers of the PSD are recruited to nascent postsynaptic sites. GFP-tagged NR1, the essential subunit of the NMDA receptor, and ProSAP1/Shank2 and ProSAP2/Shank3, scaffolding molecules thought to reside at deeper layers of the PSD, were recruited to new synaptic sites in gradual manner, with no obvious involvement of discernible discrete transport particles. The recruitment kinetics of these three PSD molecules were remarkably similar, which may indicate that PSD assembly rate is governed by a common upstream rate-limiting process. In contrast, the presynaptic active zone (AZ) molecule Bassoon was observed to be recruited to new presynaptic sites by means of a small number of mobile packets, in full agreement with previous studies. These findings indicate that the assembly processes of PSDs and AZs may be fundamentally different.

Local sharing as a predominant determinant of synaptic matrix molecular dynamics.

Recent studies suggest that central nervous system synapses can persist for weeks, months, perhaps lifetimes, yet little is known as to how synapses maintain their structural and functional characteristics for so long. As a step toward a better understanding of synaptic maintenance we examined the loss, redistribution, reincorporation, and replenishment dynamics of Synapsin I and ProSAP2/Shank3, prominent presynaptic and postsynaptic matrix molecules, respectively. Fluorescence recovery after photobleaching and photoactivation experiments revealed that both molecules are continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of minutes to hours. Exchange rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation, were accelerated by stimulation, and greatly exceeded rates of replenishment from somatic sources. These findings indicate that the dynamics of key synaptic matrix molecules may be dominated by local protein exchange and redistribution, whereas protein synthesis and degradation serve to maintain and regulate the sizes of local, shared pools of these proteins.

The dynamics of SAP90/PSD-95 recruitment to new synaptic junctions

SAP90/PSD-95 is thought to be a central organizer of the glutamatergic synapse postsynaptic reception apparatus. To assess its potential role during glutamatergic synapse formation, we used GFP-tagged SAP90/PSD-95, time lapse confocal microscopy, and cultured hippocampal neurons to determine its dynamic recruitment into new synaptic junctions. We report that new SAP90/PSD-95 clusters first appeared at new axodendritic contact sites within 20-60 min of contact establishment. SAP90/PSD-95 clustering was rapid, with kinetics that fit a single exponential with a mean time constant of approximately 23 min. Most new SAP90/PSD-95 clusters were found juxtaposed to functional presynaptic boutons as determined by labeling with FM 4-64. No evidence was found for the existence of discrete transport particles similar to those previously reported to mediate presynaptic active zone cytoskeleton assembly. Instead, we found that SAP90/PSD-95 is recruited to nascent synapses from a diffuse dendritic cytoplasmic pool. Our findings show that SAP90/PSD-95 is recruited to nascent synaptic junctions early during the assembly process and indicate that its assimilation is fundamentally different from that of presynaptic active zone components.

Long-term relationships between synaptic tenacity, synaptic remodeling, and network activity.

Long term time-lapse imaging reveals that individual synapses undergo significant structural remodeling not only when driven by activity, but also when network activity is absent, raising questions about how reliably individual synapses maintain connections.

Assembly of Active Zone Precursor Vesicles OBLIGATORY TRAFFICKING OF PRESYNAPTIC CYTOMATRIX PROTEINS BASSOON AND PICCOLO VIA A TRANS-GOLGI COMPARTMENT

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.