Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with AMPARs in artificial expression systems, but it is unknown whether Shisa7 has a functional role in glutamatergic synapses. We show that Shisa7 physically interacts with synaptic AMPARs in mouse hippocampus. Shisa7 gene deletion resulted in faster AMPAR currents in CA1 synapses, without affecting its synaptic expression. Shisa7 KO mice showed reduced initiation and maintenance of long-term potentiation of glutamatergic synapses. In line with this, Shisa7 KO mice showed a specific deficit in contextual fear memory, both short-term and long-term after conditioning, whereas auditory fear memory and anxiety-related behavior were normal. Thus, Shisa7 is a bona-fide AMPAR modulatory protein affecting channel kinetics of AMPARs, necessary for synaptic hippocampal plasticity, and memory recall.

Suspension TRAPping filter (sTRAP) is an attractive sample preparation method for proteomics studies. The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter. After trypsin digestion, peptides are analyzed directly by LC-MS. Here, we demonstrated the use of a low-cost plasmid DNA micro-spin column for the sTRAP sample preparation of a dilution series of a synapse-enriched sample with a range of 10–0.3 µg. With 120 ng tryptic peptides loaded onto the Evosep LC system coupled to timsTOF Pro 2 mass spectrometer, we identified 5700 protein groups with 4% coefficient of variation (CoV). Comparing other sample preparation protocols, such as the in-gel digestion and the commercial Protifi S-TRAP with the plasmid DNA micro-spin column, the last is superior in both protein and peptide identification numbers and CoV. We applied sTRAP for the analysis of the hippocampal proteome from the 5xFAD mouse model of Alzheimer’s disease and their wildtype littermates, and revealed 121 up- and 54 down-regulated proteins. Protein changes in the mutant mice point to the alteration of processes related to the immune system and Amyloid aggregation, which correlates well with the known major Alzheimer’s-disease-related pathology. Data are available via ProteomeXchange with the identifier PXD041045.

Glycine receptors (GlyRs) are typically known for mediating inhibitory synaptic transmission within the spinal cord and brainstem, but they also have key roles in embryonic brain development, learning/memory, inflammatory pain sensitization, and rhythmic breathing. GlyR dysfunction has been implicated in multiple neurological disease states, including startle disease (GlyR α1β) and neurodevelopmental disorders (NDDs) including autism spectrum disorder (ASD), intellectual disability (ID), developmental delay (DD) and epilepsy (GlyR α2). However, GlyRs do not operate in isolation but depend upon stable and transient protein–protein interactions (PPIs) that influence synaptic localization, homeostasis, signaling pathways, and receptor function. Despite the affinity purification of GlyRs using the antagonist strychnine over four decades ago, we still have much to learn about native GlyR stoichiometry and accessory proteins. In contrast to other neurotransmitter receptors, < 20 potential GlyR interactors have been identified to date. These include some well‐known proteins that are vital to inhibitory synapse function, such as the postsynaptic scaffolding protein gephyrin and the RhoGEF collybistin. However, the majority of known interactors either bind to the GlyR α1 and β subunits, or the binding partner in the GlyR complex is unknown. Several potential GlyR interactors are not found at inhibitory synapses and/or have no clear functional role. Moreover, other GlyR interactors are secondary interactors that bind indirectly, for example, via gephyrin. In this review, we provide a critical evaluation of known GlyR interacting proteins and methodological limitations to date. We also provide a road map for the use of innovative and emerging interaction proteomic techniques that will unlock the GlyR interactome. With the emergence of disease‐associated missense mutations in the α1, α2 and β subunit intracellular domains in startle disease and NDDs, understanding the identity and roles of GlyR accessory proteins is vital in understanding GlyR function and dysfunction in health and disease. image

Neurons release neurotransmitters from synaptic vesicles (SVs) and neuropeptides from dense-core vesicles (DCVs). The presynaptic proteins RIM and MUNC13 play key roles in both pathways. It remains unclear how DCVs are targeted to release sites and whether RIM and MUNC13 are involved in this process. Here, we show that three membrane-binding domains in RIM and MUNC13 regulate DCV exocytosis differently from SV exocytosis. Using neuropeptide secretion assays with single-vesicle resolution and peptidomics analysis of endogenous neuropeptide release in MUNC13/RIM null neurons, we demonstrate that MUNC13 is essential for DCV exocytosis. The RIM N terminus prevents MUNC13 degradation via the proteasome, and inhibiting proteasomal degradation partially rescues DCV exocytosis in RIM’s absence. Unlike SV exocytosis, the PIP2-binding RIM C2B domain and MUNC13 C1-C2B polybasic face are redundant for DCV exocytosis, while the lipid-binding MUNC13 C2C domain is crucial. These results show that RIM and MUNC13 synergistically regulate DCV exocytosis through membrane interactions and reveal new mechanistic differences between SV and DCV exocytosis.

Astrocytes are increasingly implicated in the pathophysiology of schizophrenia (SCZ), yet how astrocytic dysfunction contributes to disease-relevant neuronal abnormalities remains unclear. Here, we used mass spectrometry–based proteomics to profile lysates (proteome) and secreted proteins (secretome) from iPSC-derived astrocytes originating from 9 SCZ patients and 8 healthy controls. Compartment-specific analyses showed that lysates were enriched for mitochondrial and nuclear pathways, whereas astrocyte-conditioned media (ACM) were enriched for extracellular matrix (ECM) and vesicle-associated proteins. Differential expression analysis revealed minimal overlap between dysregulated proteins in lysates and ACM, suggesting modality-specific effects of SCZ-associated donor background. Interestingly, ECM proteins and key secreted cues involved in synaptic development, including MFGE8 and SEMA3C, were selectively reduced in SCZ ACM, whereas RNA-processing proteins were aberrantly increased. This is in line with previously reported microRNA enrichment in extracellular vesicles (EV) derived from SCZ patients. Gene set analyses further identified the alteration in secretion and nuclear processes as well as the potential involvement of autophagy-dependent release mechanism in SCZ astrocytes. Together, these findings suggest disrupted astrocytic protein homeostasis and extracellular signalling in SCZ iPSC-derived astrocytes, providing mechanistic insight into astrocyte-mediated contributions to synaptic and circuit deficits in the disorder.

Enhanced synaptic wiring onto sparsely distributed memory engram cells supports contextual memory storage and recall, with negative-valence memories exhibiting greater salience. Protein-level adaptations of input-specific synaptic connectivity onto hippocampal CA1 engram cells, however, remain largely unexplored. By combining spatiotemporally restricted synapse labeling, sorting, and mass spectrometry, we generated a discovery-oriented proteomic dataset from samples enriched for CA1 engram cell synapses receiving CA3 input 72 hours after neutral context exploration or aversive contextual fear conditioning. Differential analysis relative to an unlabeled comparator identified protein expression signatures associated with synapse structure, efficacy, and strength in CA1 engram cell synapses. Aversive learning induced predominantly postsynaptic protein expression patterns, whereas neutral context skewed toward presynaptic changes. This differential engram cell synapse proteome was enriched for genes linked to cognitive genetic traits and related disorders. Together, these data provide a hypothesis-generating resource for investigations into the molecular basis of contextual memory at the level of the synapse.