SNARE Function Research Articles

Abstract B112: SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response

Abstract The goal of this study was to investigate whether SNARE proteins are necessary for immunoglobulin (Ig) free light chain (FLC) secretion, thus representing a yet unexplored therapeutic target in AL amyloidosis (AL) and Multiple myeloma (MM). Both these disorders are incurable, and in the former, pathogenesis is invariably related to deposition of Ig FLC fibrils in target organs. The molecular mechanisms underlying FLC folding/secretion have not been studied, and no therapies directly target this intrinsic vulnerability. We hypothesize that FLC secretion is at least in part mediated by SNARE proteins, and their inhibition would block FLC exocytosis, causing retention of FLC-loaded vesicles, leading to activation of a terminal unfolded protein response (UPR) and apoptosis. We therefore hypothesized that novel, effective targets for MM and AL therapy could be identified within SNAREs. As a tool to study SNARE function, we stably expressed 7 distinct, tetracycline-inducible botulinum neurotoxin (BoNT) serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1, ALMC2) and MM (U266, KMS11) cell lines. To monitor BoNT expression, a bicistronic lentiviral vector with a P2A-GFP was used. Upon doxycycline administration, we performed growth competition assay in polyclonally transduced cells and assessed kinetics of GFP expression over time and apoptosis via flow cytometry in monoclones. Western blot (WB) was used to assess cleavage of SNAREs upon expression of each BoNT. We used WB and ELISA to assess FLC secretion on a per-cell base upon BoNT expression. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and CRISPR-Cas9 gene editing to validate putative BoNT targets. By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SYNTAXIN4, and SNAP23 as the top expressed SNARE and confirmed their high expression in a panel of AL and MM cell lines. Expression of all BoNT serotypes, except BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for extent and rapidity of cytotoxicity. Concurrent cleavage of SNAP23, VAMP3, and SYNTAXIN4 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not BoNT B, blocked FLC secretion and triggered the PERK-mediated terminal UPR with induction of CHOP and GADD34 followed by massive apoptosis. We identified SNAP23 as an interacting partner of cytotoxic BoNT F, but not BoNT B. Single SNAP23 KO did not completely recapitulate cytotoxic BoNT phenotype, as it was only partially cytotoxic. Based on our preliminary data, we hypothesize SYNTAXIN4 and VAMP3 to be synthetic lethal with SNAP23. Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. Using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosis, paving the way for the design of novel therapeutics. Citation Format: Emre Karayol, Maria Moscvin, Tianzeng Chen, Peter G. Czarnecki, Annamaria Gulla, Kenneth C. Anderson, Giada Bianchi. SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B112.

A tyrosine phospho-switch within the Longin domain of VAMP721 modulates SNARE functionality.

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.

Partial Retraction of: The EMBO Journal (2010) 29: 3607-3620. DOI: 10.1038/emboj.2010.237 | Published online 24 September 2010 Journal statement The journal contacted the authors in February 2022 about potential image insertions and duplications in Fig 4A and 4E. In the absence of source data, the authors are retracting Fig 4A, the lower panel of Fig 4E (LAMP1 immunoblot), and the following statements in the text that rely on these data: "Quantitative analysis showed that the percentage of Flotillin-1 associated with DRMs was increased in LSD endolysosomal membranes (Figure4A), indicating an increased amount of cholesterol-enriched regions in these membrane samples." "LAMP1 also displayed a similar distribution profile in WT and LSD cells (Figure4E)". Author statement The authors could not verify the aberrations in panel A of Fig 4 and the lower immunoblot (LAMP1) of 4E because the original source data are no longer available (12 years after publication, which is beyond the institute's 10-year data retention policy). The authors wish to clarify that the main conclusions of the paper are not affected by the retraction of Figurepanels 4A and 4E for the following reasons: Figurepanel 4A supports the observation that there are increased cholesterol-enhanced regions in LSD samples. This finding is also supported by data provided in figs 4B, 4C and 4D. Figurepanel 4E: The LAMP1 blot in Fig 4E shows that the distribution of protein normally excluded from DRMs is not altered between Wt and LSD samples. This result is also supported by the upper blot in this panel (Transferrin receptor). The authors apologize for these errors and agree with this corrigendum; no response could be obtained from AL.

Rerouting trafficking circuits through posttranslational SNARE modifications.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are membrane-associated trafficking proteins that confer identity to lipid membranes and facilitate membrane fusion. These functions are achieved through the complexing of Q-SNAREs with a specific cognate target R-SNARE, leading to the fusion of their associated membranes. These SNARE complexes then dissociate so that the Q-SNAREs and R-SNAREs can repeat this cycle. Whilst the basic function of SNAREs has been long appreciated, it is becoming increasingly clear that the cell can control the localisation and function of SNARE proteins through posttranslational modifications (PTMs), such as phosphorylation and ubiquitylation. Whilst numerous proteomic methods have shown that SNARE proteins are subject to these modifications, little is known about how these modifications regulate SNARE function. However, it is clear that these PTMs provide cells with an incredible functional plasticity; SNARE PTMs enable cells to respond to an ever-changing extracellular environment through the rerouting of membrane traffic. In this Review, we summarise key findings regarding SNARE regulation by PTMs and discuss how these modifications reprogramme membrane trafficking pathways.

The Dissection of SNAREs Reveals Key Factors for Vesicular Trafficking to the Endosome-like Compartment and Apicoplast via the Secretory System in Toxoplasma gondii.

ABSTRACTVesicular trafficking is a fundamental cellular process involved in material transport in eukaryotes, but the diversity of the intracellular compartments has prevented researchers from obtaining a clear understanding of the specific functions of vesicular trafficking factors, including SNAREs, tethers, and Rab GTPases, in Apicomplexa. In this study, we analyzed the localization of SNAREs and investigated their roles in vesicular trafficking in Toxoplasma gondii. Our results revealed the specific localizations of SNAREs in the endoplasmic reticulum (ER) (T. gondii Stx18 [TgStx18] and TgStx19), Golgi stacks (TgGS27), and endosome-like compartment (TgStx10 and TgStx12). The conditional ablation of ER- and Golgi-residing SNAREs caused severe defects in the secretory system. Most importantly, we found an R-SNARE (TgVAMP4-2) that is targeted to the apicoplast; to our knowledge, this work provides the first information showing a SNARE protein on endosymbiotic organelles and functioning in vesicular trafficking in eukaryotes. Conditional knockout of TgVAMP4-2 blocked the entrance of TgCPN60, TgACP, TgATrx2, and TgATrx1 into the apicoplast and interfered with the targeting of TgAPT1 and TgFtsH1 to the outermost membrane of the apicoplast. Together, our findings revealed the functions of SNAREs in the secretory system and the transport of nucleus-encoded proteins to an endosymbiotic organelle in a model organism of Apicomplexa.

ER-PM Contact Sites - SNARING Actors in Emerging Functions.

The compartmentalisation achieved by confining cytoplasm into membrane-enclosed organelles in eukaryotic cells is essential for maintaining vital functions including ATP production, synthetic and degradative pathways. While intracellular organelles are highly specialised in these functions, the restricting membranes also impede exchange of molecules responsible for the synchronised and responsive cellular activities. The initial identification of contact sites between the ER and plasma membrane (PM) provided a potential candidate structure for communication between organelles without mixing by fusion. Over the past decades, research has revealed a far broader picture of the events. Membrane contact sites (MCSs) have been recognized as increasingly important actors in cell differentiation, plasticity and maintenance, and, upon dysfunction, responsible for pathological conditions such as cancer and neurodegenerative diseases. Present in multiple organelles and cell types, MCSs promote transport of lipids and Ca2+ homoeostasis, with a range of associated protein families. Interestingly, each MCS displays a unique molecular signature, adapted to organelle functions. This review will explore the literature describing the molecular components and interactions taking place at ER-PM contact sites, their functions, and implications in eukaryotic cells, particularly neurons, with emphasis on lipid transfer proteins and emerging function of SNAREs.

New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System.

Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.

An In Vitro Assay of Trans-SNARE Complex Formation During Yeast Vacuole Fusion Using Epitope Tag-Free SNAREs.

SNARE complexes assembled between fusing membranes (in trans) are the core machinery driving lipid bilayer merger. Thus, an assay monitoring the formation of these trans-SNARE complexes is essential for SNARE-mediated membrane fusion studies. Homotypic yeast vacuole fusion is an important model system for such studies. Although several assays measuring trans-SNARE complex formation are available to study yeast vacuole fusion, most use SNAREs conjugated with epitope tags, which may affect the function of SNAREs or even the formation of trans-SNARE complexes. Here, I describe an assay for trans-SNARE complex formation during yeast vacuole fusion that does not require epitope-tagged SNAREs.

SNARE dynamics during melanosome maturation.

Historically, studies on the maturation and intracellular transport of melanosomes in melanocytes have greatly contributed to elucidating the general mechanisms of intracellular transport in many different types of mammalian cells. During melanosome maturation, melanosome cargoes including melanogenic enzymes (e.g. tyrosinase) are transported from endosomes to immature melanosomes by membrane trafficking, which must require a membrane fusion process likely regulated by SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors]. In the present study, we review the literature concerning the expression and function of SNAREs (e.g. v-SNARE vesicle-associated membrane protein 7 and t-SNAREs syntaxin-3/13 and synaptosomal-associated protein-23) in melanocytes, especially in regard to the fusion process in which melanosome cargoes are finally delivered to immature melanosomes. We also describe the recent discovery of the SNARE recycling system on mature melanosomes in melanocytes. Such SNARE dynamics, especially the SNARE recycling system, on melanosomes will be useful in understanding as yet unidentified SNARE dynamics on other organelles.

The tomosyn homologue, Sro7, is a direct effector of the Rab GTPase, Sec4, in post-Golgi vesicle tethering.

The tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7’s ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7’s functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.

Rab5 is critical for SNAP23 regulated granule-granule fusion during compound exocytosis

Compound exocytosis is considered the most massive mode of exocytosis, during which the membranes of secretory granules (SGs) fuse with each other to form a channel through which the entire contents of their granules is released. The underlying mechanisms of compound exocytosis remain largely unresolved. Here we show that the small GTPase Rab5, a known regulator of endocytosis, is pivotal for compound exocytosis in mast cells. Silencing of Rab5 shifts receptor-triggered secretion from a compound to a full exocytosis mode, in which SGs individually fuse with the plasma membrane. Moreover, we show that Rab5 is essential for FcεRI-triggered association of the SNARE protein SNAP23 with the SGs. Direct evidence is provided for SNAP23 involvement in homotypic SG fusion that occurs in the activated cells. Finally, we show that this fusion event is prevented by inhibition of the IKKβ2 kinase, however, neither a phosphorylation-deficient nor a phosphomimetic mutant of SNAP23 can mediate homotypic SG fusion in triggered cells. Taken together our findings identify Rab5 as a heretofore-unrecognized regulator of compound exocytosis that is essential for SNAP23-mediated granule-granule fusion. Our results also implicate phosphorylation cycles in controlling SNAP23 SNARE function in homotypic SG fusion.

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion.

Functional assays for the assessment of the pathogenicity of variants of GOSR2, an ER-to-Golgi SNARE involved in progressive myoclonus epilepsies.

ABSTRACTProgressive myoclonus epilepsies (PMEs) are inherited disorders characterized by myoclonus, generalized tonic-clonic seizures, and ataxia. One of the genes that is associated with PME is the ER-to-Golgi Qb-SNARE GOSR2, which forms a SNARE complex with syntaxin-5, Bet1 and Sec22b. Most PME patients are homo­zygous for a p.Gly144Trp mutation and develop similar clinical presentations. Recently, a patient who was compound heterozygous for p.Gly144Trp and a previously unseen p.Lys164del mutation was identified. Because this patient presented with a milder disease phenotype, we hypothesized that the p.Lys164del mutation may be less severe compared to p.Gly144Trp. To characterize the effect of the p.Gly144Trp and p.Lys164del mutations, both of which are present in the SNARE motif of GOSR2, we examined the corresponding mutations in the yeast ortholog Bos1. Yeasts expressing the orthologous mutants in Bos1 showed impaired growth, suggesting a partial loss of function, which was more severe for the Bos1 p.Gly176Trp mutation. Using anisotropy and gel filtration, we report that Bos1 p.Gly176Trp and p.Arg196del are capable of complex formation, but with partly reduced activity. Molecular dynamics (MD) simulations showed that the hydrophobic core, which triggers SNARE complex formation, is compromised due to the glycine-to-tryptophan substitution in both GOSR2 and Bos1. In contrast, the deletion of residue p.Lys164 (or p.Arg196del in Bos1) interferes with the formation of hydrogen bonds between GOSR2 and syntaxin-5. Despite these perturbations, all SNARE complexes stayed intact during longer simulations. Thus, our data suggest that the milder course of disease in compound heterozygous PME is due to less severe impairment of the SNARE function.

Balancing Order and Disorder in Neurodegeneration and Neurotransmission

Neurons communicate primarily via the release of chemical neurotransmitters from presynaptic nerve terminals and their detection post-synaptically. Thus, the process of synaptic vesicle exocytosis is at the heart of neuronal function and cognition. Intriguingly, the proteins that play the key role in the fusion of vesicles with membranes, the SNARE proteins, are IDPs, and a number of the additional factors that regulate neuronal SNARE function are also intrinsically disordered, including the proteins alpha-synuclein and complexin. Our work has helped to establish the mechanisms by which disorder-to-order transitions in both complexin and alpha-synuclein contribute to the functional aspects of these proteins, and in the case of alpha-synuclein, to their role in neurodegenerative disease as well. Recent progress in these areas will be described.

Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers

AbstractSNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid‐conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA–lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base‐pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE‐mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA–lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.

Accelerating SNARE-Mediated Membrane Fusion by DNA-Lipid Tethers.

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid-conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA-lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base-pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE-mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA-lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.

Ubiquitin-Synaptobrevin Fusion Protein Causes Degeneration of Presynaptic Motor Terminals in Mice.

Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (Ub(G76V)-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration.

SNAREs Controlling Vesicular Release of BDNF and Development of Callosal Axons.

At presynaptic active zones, exocytosis of neurotransmitter vesicles (SVs) is driven by SNARE complexes that recruit Syb2 and SNAP25. However, it remains unknown which SNAREs promote the secretion of neuronal proteins, including those essential for circuit development and experience-dependent plasticity. Here we demonstrate that Syb2 and SNAP25 mediate the vesicular release of BDNF in axons and dendrites of cortical neurons, suggesting these SNAREs act in multiple spatially segregated secretory pathways. Remarkably, axonal secretion of BDNF is also strongly regulated by SNAP47, which interacts with SNAP25 but appears to be dispensable for exocytosis of SVs. Cell-autonomous ablation of SNAP47 disrupts the layer-specific branching of callosal axons of projection cortical neurons in vivo, and this phenotype is recapitulated by ablation of BDNF or its receptor, TrkB. Our results provide insights into the molecular mechanisms of protein secretion, and they define the functions of SNAREs in BDNF signaling and regulation of neuronal connectivity.

Organelle and proteome quality control mechanisms: how cells are able to keep calm and carry on

Organelle and proteome quality control mechanisms: how cells are able to keep calm and carry on

Cognitive Enhancing Treatment with a PPARγ Agonist Normalizes Dentate Granule Cell Presynaptic Function in Tg2576 APP Mice

Hippocampal network hyperexcitability is considered an early indicator of Alzheimer's disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I-O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I-O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.