Sergio Leal-Ortiz

v-SNARE Composition Distinguishes Synaptic Vesicle Pools

Synaptic vesicles belong to two distinct pools, a recycling pool responsible for the evoked release of neurotransmitter and a resting pool unresponsive to stimulation. The uniform appearance of synaptic vesicles has suggested that differences in location or cytoskeletal association account for these differences in function. We now find that the v-SNARE tetanus toxin-insensitive vesicle-associated membrane protein (VAMP7) differs from other synaptic vesicle proteins in its distribution to the two pools, providing evidence that they differ in molecular composition. We also find that both resting and recycling pools undergo spontaneous release, and when activated by deletion of the longin domain, VAMP7 influences the properties of release. Further, the endocytosis that follows evoked and spontaneous release differs in mechanism, and specific sequences confer targeting to the different vesicle pools. The results suggest that different endocytic mechanisms generate synaptic vesicles with different proteins that can endow the vesicles with distinct properties.

Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis

Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II–dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.

Nanostraw–Electroporation System for Highly Efficient Intracellular Delivery and Transfection

Nondestructive introduction of genes, proteins, and small molecules into mammalian cells with high efficiency is a challenging, yet critical, process. Here we demonstrate a simple nanoelectroporation platform to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield cotransfection and sequential transfection.

Synaptic SAP97 Isoforms Regulate AMPA Receptor Dynamics and Access to Presynaptic Glutamate

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, attributable to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated α-isoform to the postsynaptic density (PSD) and the L27 domain-containing β-isoform primarily to non-PSD, perisynaptic regions. Consequently, α- and βSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs and, hence, their responsiveness to presynaptically released glutamate.

Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2.

SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli.

Rapid Assembly of Functional Presynaptic Boutons Triggered by Adhesive Contacts

CNS synapse assembly typically follows after stable contacts between “appropriate” axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.

Quantification of nanowire penetration into living cells

High-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.

Bassoon and Piccolo maintain synapse integrity by regulating protein ubiquitination and degradation.

The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis.

Piccolo Regulates the Dynamic Assembly of Presynaptic F-Actin

Filamentous (F)-actin is a known regulator of the synaptic vesicle (SV) cycle, with roles in SV mobilization, fusion, and endocytosis. However, the molecular pathways that regulate its dynamic assembly within presynaptic boutons remain unclear. In this study, we have used shRNA-mediated knockdown to demonstrate that Piccolo, a multidomain protein of the active zone cytomatrix, is a key regulator of presynaptic F-actin assembly. Boutons lacking Piccolo exhibit enhanced activity-dependent Synapsin1a dispersion and SV exocytosis, and reduced F-actin polymerization and CaMKII recruitment. These phenotypes are rescued by stabilizing F-actin filaments and mimicked by knocking down Profilin2, another regulator of presynaptic F-actin assembly. Importantly, we find that mice with a targeted deletion of exon 14 from the Pclo gene, reported to lack >95% of Piccolo, continue to express multiple Piccolo isoforms. Furthermore, neurons cultured from these mice exhibit no defects in presynaptic F-actin assembly due to the expression of these isoforms at presynaptic boutons. These data reveal that Piccolo regulates neurotransmitter release by facilitating activity-dependent F-actin assembly and the dynamic recruitment of key signaling molecules into presynaptic boutons, and highlight the need for new genetic models with which to study Piccolo loss of function.

Formation of Golgi-Derived Active Zone Precursor Vesicles

Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1α associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.