Biology subjects

Blanpied, T. A.

Publications and source records attributed to Blanpied, T. A..

15 recordsLinked to original sources

bioRxiv2026

Nanoarchitecture of the presynaptic cell adhesion molecule PTPσ in relation to prospective vesicle fusion sites and postsynaptic NMDA receptors

Protein organization at individual synapses tunes synaptic function. Presynaptic release-regulating proteins and postsynaptic receptors each concentrate into subsynaptic nanoclusters, and their precise alignment across the cleft into nanocolumns can maximize receptor activation following vesicle fusion. However, both the molecular organization defining presynaptic fusion sites and the mechanisms by which they are spatially coordinated with postsynaptic complexes remains unclear. Presynaptic transmembrane cell adhesion molecules may mediate both processes as they could simultaneously anchor cytosolic fusion machinery to the presynaptic membrane and bridge the cleft to coordinate PSD organization. The LAR-RPTP family member PTP{sigma} is an important possibility, as its trans-synaptic interactors that bind PSD-95 are associated with NMDAR dysfunction, suggesting PTP{sigma} may coordinate NMDAR positioning. However, PTP{sigma}'s subsynaptic organization has not been determined. Here, we investigate the cellular expression and subsynaptic nano-organization of endogenous PTP{sigma} in cultured rat hippocampal neurons. We find that PTP{sigma} is enriched at most excitatory synapses, but unexpectedly, PTP{sigma} nanoclusters do not colocalize with the vesicle priming protein Munc13-1. Instead, they adopt a stereotyped position adjacent to this machinery, without preferential association with sites in nanocolumns. PTP{sigma} nanoclusters also lie offset from postsynaptic NMDA receptors, mirroring their relationship to vesicle fusion sites, yet the presence of PTP{sigma} at a nanocolumn does not predict NMDAR abundance. Together, these findings suggest that PTP{sigma} does not directly coordinate release site - NMDAR alignment but instead occupies defined nanoscale positions adjacent to vesicle fusion sites, potentially helping to shape or stabilize the vesicle priming landscape of the active zone.

neuroscience↗

bioRxiv2026

Btbd11 regulates glutamatergic synapse organization in GABAergic inhibitory interneurons

The mechanisms that underlie glutamatergic synapse organization and function in GABAergic inhibitory interneurons (INs) are not well described, despite evidence that impaired glutamatergic excitation of INs is implicated in psychiatric disorders such as schizophrenia and anxiety. Glutamatergic synapses received by INs have unique basal transmission properties and exhibit distinct synaptic plasticity compared to those received by excitatory neurons, likely due to cell-type specific differences in postsynaptic density (PSD) composition and maintenance mechanisms. In the present study, we show that the interneuron-specific protein Btbd11 regulates excitatory synapse transmission in hippocampal interneurons through promotion of phase separation and support of postsynaptic nanoarchitecture. Btbd11 forms a phase separated protein complex with Psd-95 and TARP{gamma}2 and impacts the stability of TARP{gamma}2 and GluA1 within glutamatergic IN synapses in an expression- and phase separation-dependent manner. Using super resolution imaging, we show that Btbd11 displays nanoscale clustering properties within IN synapses that correlate with Psd-95 nanostructure. Furthermore, genetic deletion of Btbd11 decreases PSD protein expression, reduces synapse size, and disrupts Psd-95 nanocluster organization. These effects manifest as a drastic reduction in glutamatergic synaptic transmission onto INs when Btbd11 is deleted. Together, these data provide insights into a novel cell type-specific synaptic regulatory mechanism in an understudied synapse population.

neuroscience↗

bioRxiv2026

Structural Components for Calcitonin Gene-Related Peptide Signaling to Oligodendrocyte Precursor Cells

Oligodendrocyte precursor cells (OPCs) are unique glial cells that communicate bidirectionally with neurons. Neuronal inputs drive various OPC behaviors, including proliferation and differentiation, immunomodulation, blood brain barrier regulation, synapse engulfment and axonal remodeling. OPCs are implicated in numerous stress and pain conditions, where their involvement is likely driven by neuronal activity (ie. neurotransmitter and neuropeptide signaling). One neuropeptide causally involved in chronic pain and stress conditions is calcitonin gene-related peptide (CGRP). Here, we tested the hypothesis that OPCs receive direct inputs from CGRP-containing neurons in the adult brain. Using RNAscope, immunofluorescence and analysis of single-cell datasets, we find that OPCs express receptors for CGRP and we identify close spatial contacts between CGRP and OPCs, with nearly half of CGRP puncta occurring within 1 {micro}m of an OPC. Some of these contacts appear to be synaptic, with CGRP-OPC contacts colocalizing with the presynaptic protein Bassoon and the postsynaptic protein PSD-95. This work suggests the presence of both diffuse and more direct forms of CGRP signaling to OPCs, raising the importance of future experiments to identify both the mode of CGRP release onto OPCs and the functional effects of these different contact types.

neuroscience↗

bioRxiv2025

Lysosomal multi-omics reveals altered sphingolipid catabolism as driver of lysosomal dysfunction in the aging brain.

Recent data indicate that lipid composition has profound influence on the brain function and that changes in lipid homeostasis affect brain aging and predisposition to neurodegenerative diseases. Lipids dynamically reside in multiple intracellular locations and their organellar distribution is important for specific interactions and biological function. During brain aging lipid changes have been specifically noted in lysosomes, but the identity of the accumulated lipids, their interactions with other biomolecules such as proteins, and their functional relevance have not been characterized. We used mass spectrometry (MS) to assess longitudinal changes in the lipidome and proteome of lysosomes isolated from the mouse cortex, from the age of 3- to 24-months. Our statistical and machine learning analyses identified two factors demonstrating predictive power for age and differences in both lipids and proteins. Of these, factor 1 was the best predictor of sample age. Factor 1 lipids with the highest feature importance included multiple species of hexosylceramides (HexCer) and their sulfonated derivatives, sulfatides (SHexCer), all of which increased with age. Increased factor 1 proteins included myelin proteins, select sphingolipid catabolism enzymes and proteins associated with lysosomal storage diseases. Our analyses suggested that mechanisms underlying factor 1 encompass the combination of an age-dependent increase in lysosomal delivery of myelin components and alterations in lysosomal sphingolipid catabolism favoring degradation of sphingomyelin over HexCer. The overall age-related lysosomal changes resembled those observed in lysosomal storage diseases, particularly Gaucher disease, where accumulation of HexCer species is associated with lysosomal dysfunction. To corroborate factor 1 predictions, we employed a combination of biochemical, imaging and flow cytometry approaches, which confirmed alterations in sphingolipid catabolism and lysosomal accumulation of myelin components. These changes were associated with age-related alteration in lysosomal morphology, lysosomal dysfunction and inhibition of autophagy in both neurons and microglia. Our findings indicate that factors contributing to lysosomal aging resemble those observed in lysosomal storage diseases and underscore the significance of organelle-specific analyses for dissecting mechanisms contributing to brain aging.

neuroscience↗

bioRxiv2024

Dissecting the functional heterogeneity of glutamatergic synapses with high-throughput optical physiology

Fluorescent reporters for glutamate release and postsynaptic Ca2+ signaling are essential tools for quantifying synapse functional heterogeneity across neurons and circuits. However, leveraging these probes for neuroscience requires scalable experimental frameworks. Here, we devised a high-throughput approach to efficiently collect and analyze hundreds of optical recordings of glutaamate release activity at presynaptic boutons in cultured rat hippocampal neurons. Boutons exhibited remarkable functional heterogeneity and could be separated into multiple functional classes based on their iGluSnFR3 responses to single action potentials, paired stimuli, and synaptic parameters derived from mean-variance analysis. Finally, we developed a novel all-optical assay of pre- and postsynaptic glutamatergic synapse function. We deployed iGluSnFR3 with a red-shifted, postsynaptically-targeted Ca2+ sensor, enabling direct imaging and analysis of NMDA receptor-mediated synaptic transmission at large numbers of dendritic spines. This work enables direct observation of the flow of information at single synapses and should speed detailed investigations of synaptic functional heterogeneity.

neuroscience↗

bioRxiv2024

Large donor CRISPR for whole-CDS replacement of cell adhesion molecule LRRTM2

The cell adhesion molecule LRRTM2 is crucial for synapse development and function. However, our understanding of its endogenous trafficking has been limited due to difficulties in manipulating its coding sequence (CDS) using standard genome editing techniques. We instead replaced the whole LRRTM2 CDS by adapting the recent CRISPR method Targeted Knock-in using Two Guides (TKIT), enabling complete control of LRRTM2. In primary rat hippocampal cultures, N-terminally tagged, endogenous LRRTM2 was found in 80% of synapses, and synaptic LRRTM2 content correlated with PSD-95 and AMPAR levels. LRRTM2 was also enriched with AMPARs outside synapses, demonstrating the sensitivity of this method to detect relevant new biology. Finally, we leveraged total genomic control to increase the synaptic levels of LRRTM2 via simultaneous mutation of its C-terminal domain, which did not correspondingly increase AMPAR enrichment. The coding region of thousands of genes span lengths suitable for whole-CDS replacement, suggesting this simple approach will enable straightforward structure-function analysis in diverse cellular systems.

neuroscience↗

bioRxiv2024

Loss of postsynaptic NMDARs drives nanoscale reorganization of Munc13-1 and PSD-95

Nanoscale protein organization within the active zone (AZ) and post-synaptic density (PSD) influences synaptic transmission. Nanoclusters of presynaptic Munc13-1 are associated with readily releasable pool size and neurotransmitter vesicle priming, while postsynaptic PSD-95 nanoclusters coordinate glutamate receptors across from release sites to control their opening probability. Nanocluster number, size, and protein density vary between synapse types and with development and plasticity, supporting a wide range of functional states at the synapse. Whether or how the receptors themselves control this critical architecture remains unclear. One prominent PSD molecular complex is the NMDA receptor (NMDAR). NMDARs coordinate several modes of signaling within synapses, giving them the potential to influence synaptic organization through direct protein interactions or through signaling. We found that loss of NMDARs results in larger synapses that contain smaller, denser, and more numerous PSD-95 nanoclusters. Intriguingly, NMDAR loss also generates retrograde reorganization of the active zone, resulting in denser, more numerous Munc13-1 nanoclusters, more of which are aligned with PSD-95 nanoclusters. Together, these changes to synaptic nanostructure predict stronger AMPA receptor-mediated transmission in the absence of NMDARs. Notably, while prolonged antagonism of NMDAR activity increases Munc13-1 density within nanoclusters, it does not fully recapitulate these trans-synaptic effects. Thus, our results confirm that NMDARs play an important role in maintaining pre- and postsynaptic nanostructure and suggest that both decreased NMDAR expression and suppressed NMDAR activity may exert distinct effects on synaptic function, yet through unique architectural mechanisms. Significance StatementSynaptic transmission is shaped by the trans-synaptic coordination of molecular ensembles required for neurotransmitter release and receptor retention, but how receptors themselves influence this critical architecture remains unclear. Using state-of-the-art super-resolution microscopy, we report that loss of NMDA receptors from excitatory synapses alters both pre- and postsynaptic nano-organizational features. Notably, pharmacological antagonism of NMDA receptors also alters presynaptic features, but without fully mimicking effects of the knockout. This suggests that both NMDA receptor activity and presence at the synapse exert retrograde influence on active zone organization. Because numerous disease and activity states decrease expression or function of NMDA receptors, our results suggest that distinct nanostructural states contribute to the unique functional status of synapses in these disorders.

neuroscience↗

bioRxiv2023

Trans-synaptic molecular context of NMDA receptor nanodomains

Tight coordination of the spatial relationships between protein complexes is required for cellular function. In neuronal synapses, many proteins responsible for neurotransmission organize into subsynaptic nanoclusters whose trans-cellular alignment modulates synaptic signal propagation. However, the spatial relationships between these proteins and NMDA receptors (NMDARs), which are required for learning and memory, remain undefined. Here, we mapped the relationship of key NMDAR subunits to reference proteins in the active zone and postsynaptic density using multiplexed super-resolution DNA-PAINT microscopy. GluN2A and GluN2B subunits formed nanoclusters with diverse configurations that, surprisingly, were not localized near presynaptic vesicle release sites marked by Munc13-1. Despite this, we found a subset of release sites was enriched with NMDARs, and modeling of glutamate release and receptor activation in measured synapses indicated this nanotopography promotes NMDAR activation. This subset of release sites was internally denser with Munc13-1, aligned with abundant PSD-95, and associated closely with specific NMDAR nanodomains. Further, NMDAR activation drove rapid reorganization of this release site/receptor relationship, suggesting a structural mechanism for tuning NMDAR-mediated synaptic transmission. This work reveals a new principle regulating NMDAR signaling and suggests that synaptic functional architecture depends on the assembly of and trans-cellular spatial relationships between multiprotein nanodomains.

neuroscience↗

bioRxiv2023

Molecular definition of distinct active zone protein machineries for Ca2+ channel clustering and synaptic vesicle priming

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin- and RIM from co- assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTP{sigma} recruits Liprin-, RIM and Munc13 into priming complexes without co- clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin- and PTP{sigma} specifically support priming site assembly.

neuroscience↗

bioRxiv2023

Distinct SAP102 and PSD-95 nano-organization defines multiple types of synaptic scaffold protein domains at single synapses

The MAGUK family of scaffold proteins plays a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. Of these scaffold proteins, SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform overlapping as well as unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later in development and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could impact how SAP102 clusters synaptic proteins and underlie its ability to perform its unique functions. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters. However, SAP102 nanoclusters were smaller and denser than PSD-95 nanoclusters across development. Additionally, only a subset of SAP102 nanoclusters co-organized with PSD-95, revealing that within individual synapses there are nanodomains that contain either one or both proteins. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions. Significance statementSAP102 and PSD-95 are two key members of the MAGUK family of synaptic scaffold proteins that are critical for synapse development, maintenance, and modification during plasticity. Because PSD-95 has a highly complex subsynaptic nanostructure that impacts synaptic function, we asked if SAP102 is similarly organized into nanoclusters at individual synapses and how it relates to PSD-95 within synapses. We found that SAP102 forms subsynaptic nanoclusters with unique properties, and that within individual synapses proteins both concentrate into overlapping nanodomains, as well as form MAGUK-specific nanodomains. This demonstrates that organization of synaptic proteins into nanoclusters is likely to be maintained within the family of MAGUK proteins and reveals potential mechanism for specializing functions within individual synapses based on scaffold protein nanodomains.

neuroscience↗

bioRxiv2023

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex[->]Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex[->]PV synapses). As in Ex[->]Ex synapses, we find that in Ex[->]PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre-and postsynaptic Ex[->]PV nanostructure. Ex[->]PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex[->]Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex[->]PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of trans-synaptic alignment in these synapse types, revealing protein alignment in Ex[->]PV synapses over a distinct range of distances compared to Ex[->]Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.

neuroscience↗

bioRxiv2021

Subsynaptic positioning of AMPARs by LRRTM2 controls synaptic strength

Recent evidence suggests that nanoorganization of proteins within synapses may control the strength of communication between neurons in the brain. The unique subsynaptic distribution of glutamate receptors, which cluster in nanoalignment with presynaptic sites of glutamate release, supports this idea. However, testing it has been difficult because mechanisms controlling subsynaptic organization remain unknown. Reasoning that transcellular interactions could position AMPA receptors, we targeted a key transsynaptic adhesion molecule implicated in controlling AMPAR number, LRRTM2, using engineered, rapid proteolysis. Severing the LRRTM2 extracellular domain led quickly to nanoscale de-clustering of AMPARs away from release sites, not prompting their escape from synapses until much later. This rapid remodeling of AMPAR position produced significant deficits in evoked, but not spontaneous, postsynaptic receptor activation. These results dissociate receptor numbers from their nanopositioning in determination of synaptic function, and support the novel concept that adhesion molecules acutely position AMPA receptors to dynamically control synaptic strength.

neuroscience↗

bioRxiv2020

Expansion Revealing: Decrowding Proteins to Unmask Invisible Brain Nanostructures

Cells and tissues are made out of nanoscale building blocks, such as proteins, organized in crowded nanostructures. We show that many biomolecules, and the nanostructures in which they are embedded, may be invisible to prior imaging techniques, due to the inaccessibility of labels (e.g., antibodies) to biomolecules embedded within such crowded structures. We developed a technology, expansion revealing (ExR), which isotropically decrowds proteins from each other, to enable their labeling. We use ExR to discover the alignment of presynaptic calcium channels with postsynaptic machinery in intact brain circuits, as well as the existence of periodic amyloid nanoclusters containing ion channel proteins in Alzheimers model mice. Thus, ExR reveals nanostructures within complex biological specimens that were not previously visualizable, and may find broad use in biology. One Sentence SummaryDe-crowding biomolecules with Expansion Revealing unmasks fundamentally new nanostructures in intact brain tissue, which remained invisible otherwise.

bioengineering↗

bioRxiv2019

Properties of individual hippocampal synapses influencing NMDA-receptor activation by spontaneous neurotransmission.

NMDA receptor (NMDAR) activation is critical for maintenance and modification of synapse strength. Specifically, NMDAR activation by spontaneous glutamate release has been shown to mediate forms of synaptic plasticity as well as synaptic development. Interestingly, there is evidence that within individual synapses each release mode may be segregated such that postsynaptically there are distinct pools of responsive receptors. In order to examine potential regulators of NMDAR activation due to spontaneous glutamate release in cultured rat hippocampal neurons, we utilized GCaMP6f imaging at single synapses in concert with confocal and super-resolution imaging. Using these single spine approaches, we found that Ca2+ entry activated by spontaneous release tends to be carried by GluN2B-NMDARs. Additionally, the amount of NMDAR activation varies greatly both between synapses and within synapses, and is unrelated to spine and synapse size, but does correlate loosely with synapse distance from the soma. Despite the critical role of spontaneous activation of NMDARs in maintaining synaptic function, their activation seems to be controlled factors other than synapse size or synapse distance from the soma. It is most likely that NMDAR activation by spontaneous release influenced variability in subsynaptic receptor position, release site position, vesicle content, and channel properties. Therefore, spontaneous activation of NMDARs appears to be regulated distinctly from other receptor types, notably AMPARs, within individual synapses.\n\nSignificance StatementUnderstanding the underlying synaptic mechanisms for learning and memory is critically important to the field of neuroscience and for human health. A key neurotransmitter receptor type involved in learning is the NMDA receptor, and exploration of its regulation is vital. In this study, we optimized optical tools to allow detailed characterization of NMDA receptor activity at single synapses, along with analysis of structural features of the imaged synapses. The amount of receptor activation is independent of the size of the synapse, but weakly dependent on synapse position within the dendritic tree. Notably, we found that NMDA receptors activated following spontaneous neurotransmitter release tend be GluN2B-containing receptors. Thus, the unique mechanisms that regulate the number and positioning of these receptors within synapses will have important consequences for control of synaptic development and signaling.

neuroscience↗

bioRxiv2019

Quantitative Analysis of Trans-synaptic Protein Alignment

Nanoscale distribution of proteins and their relative positioning within a defined subcellular region are key to their physiological functions. Thanks to the super-resolution imaging methods, especially the single-molecule localization microscopy (SMLM), mapping the three-dimensional distribution of multiple proteins has been easier and more efficient than ever. In spite of the many tools available for efficient localization detection and image rendering, it has been a challenge to quantitatively analyze the 3D distribution and relative positioning of proteins in these SMLM data. Here, using the heterogeneously distributed synaptic proteins as examples, we describe in detail a series of analytical methods including detection of nanoscale density clusters, quantification of the trans-synaptic alignment between these protein densities, and automatic enface projection and averaging. These analyses were performed within customized Matlab routines and we make the full scripts available. The concepts behind these analytical methods and the scripts can be adapted for quantitative analysis of spatial organization of other macromolecular complexes.\n\nHighlightsO_LILocalization microscopy provides sufficient data for precise quantitative analysis.\nC_LIO_LIAn algorithm to identify local density peaks within a 3D localization cluster.\nC_LIO_LINew methods for quantitative analysis of trans-synaptic proteins alignment and enrichment.\nC_LIO_LIThese algorithms can be easily adapted to analysis of other subcellular organizations.\nC_LI

neuroscience↗