Biology subjects

Schuman, E. M.

Publications and source records attributed to Schuman, E. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Mouse Neural Organoids Model the Mature In Vivo Synaptic Proteome

Neural organoids represent a promising approach for modelling neural circuits and neurological disease, yet their utility depends on whether they can recapitulate the molecular complexity of mature brain synapses. Here, we optimized a protocol to generate mature forebrain organoids from mouse embryonic stem cells and used time-resolved proteomics to define the emergence of synaptic proteins during maturation. Using fluorescence-activated synaptosome sorting coupled with mass spectrometry, we quantified the synapse-enriched proteome and found a high correlation with matched in vivo synaptic proteomes. These findings demonstrate that complex mature synaptic molecular architecture is present within an in vitro organoid environment.

neuroscience↗

bioRxiv2026

Direct interaction of ribosomes with postsynaptic proteins gives rise to a privileged local synaptic translatome

Ribosomes and thousands of mRNAs are localized near synapses to support local protein synthesis. Little is known, however, about how ribosomes are positioned and maintained in dendritic spines- the primary postsynaptic sites of excitatory neurotransmission. Here, using proximity labeling-mass spectrometry, we mapped the interactome of postsynaptic ribosomes, and discovered an unexpected interaction with AMPA receptor complex proteins. Co-immunoprecipitation and crosslinking mass spectrometry using rat cortical synaptosomes showed a direct, mRNA-independent interaction between postsynaptic proteins and intact ribosomes. Immunoprecipitation-ribosome profiling (IP-Ribo-seq) revealed not only the complete synaptic translatome but also that the AMPA receptor-associated subpopulation of synaptic ribosomes preferentially translates mRNAs encoding proteins related to post-synaptic density (PSD) scaffolding and cytoskeletal dynamics. Translation of one of the mRNA targets, Camk2a, was reduced in spines following ER sequestration of endogenous GluA1. Together, these results reveal a role for PSD proteins in positioning ribosomes near the postsynaptic membrane, providing a mechanism to couple synaptic activity with the local production of proteins needed for structural remodeling.

neuroscience↗

bioRxiv2025

Brain-wide synaptosome profiling reveals localized mRNAs that diversify synapses

Chemical synapses are the principal communication nodes of the brain, defined by their specialization and plasticity. Their diversity spans multiple levels, from the polarity and magnitude of electrophysiological responses to the proteins driving these differences. Here, we describe another layer of synapse diversity - synaptic transcriptome diversity. Using transgenic mice to fluorescently label presynapses in Camk2a-, Gad2-, DAT-, PV-, SST-, and VIP-expressing neurons, combined with fluorescence-activated synaptosome sorting, we profiled synaptic transcriptomes across five brain regions. We identified [~]4000 mRNAs enriched at synapses - some type-specific, others shared - and highlight region-specific enrichments of mRNAs encoding protein subunits or family members. We found that the abundance of synaptic mRNAs is not a passive reflection of their abundance in cellular somata, indicating active trafficking and sorting mechanisms. Integrating transcriptomic and proteomic data, we identified [~]90 genes with significantly correlated mRNA-protein ratios across regions, mainly involved in synaptic vesicle dynamics, receptor signaling, and calcium regulation, suggesting a key role for local translation in maintaining protein copy number. Although synaptic mRNAs represent only a fraction of the templates for the synaptic proteome, the synaptic transcriptome reflects the synapse diversity captured by the proteome equally well. Together, this dataset (https://syndive.org/) provides a resource for exploring how synaptic mRNA localization and local translation shape synaptic identity and function.

neuroscience↗

bioRxiv2025

Localized mRNAs and protein synthesis in cortical layer 1

Cortical layer 1 plays an essential role for brain function, integrating many streams of information and undergoing synaptic plasticity during learning. Yet, we understand little of the underlying molecular processes, and whether they are specialized to achieve layer-specific functions. Here, we show that layer 1 and its synapses are metabolically active. Using laser capture RNA-sequencing and fluorescence in situ hybridization, we report an abundance of synaptic transcripts localized to layer 1. We purified layer 1 excitatory and inhibitory synapses and characterized their enriched transcriptomes, identifying candidate proteins that can be locally synthesized to maintain and regulate synaptic transmission in layer 1. Furthermore, we find significant differences between the transcriptome of synapses in layer 1 and in deeper, somatic layers, suggesting that local translation might confer specialized function. Finally, by comparing our tissue data with the transcriptome from the hippocampal CA1 strata, we discover a strong similarity between cortical layer 1 and stratum lacunosum moleculare, suggesting that these distal layers share a common molecular environment. Together, our results establish that local protein synthesis is an important mechanism for layer 1, and provide the first comprehensive characterization of the transcripts localized to layer 1 and its synapses.

neuroscience↗

bioRxiv2025

Correlative MS Imaging for cellular identification and analysis of in situ cryo-ET

In situ cryo-electron tomography (cryo-ET) offers direct access to protein structures in their native state and environment. A central challenge for this technique is the identification and classification of imaged cells, as sample heterogeneity with respect to cell type and functional state can significantly impact the data. Currently, cells can be classified using subtomogram averaging or fluorescent labeling, which require sufficiently distinguishable macromolecules, genetic modifications, or the availability of suitable labels. In MALDI-MS Imaging (MSI) individual cells can be identified and classified reliably, making use of the distributions of phospholipid species, metabolites and small proteins with characteristic profiles for specific cell types. Here we present an integrated pipeline combining MSI and in situ cryo-ET for single-cell identification and classification. Our approach enables MSI data acquisition directly from EM grids and can be used on any eukaryotic single-cell cryo-ET sample without prior modification in less than 36 h. We apply this workflow to describe cell-to-cell heterogeneity in cultured HeLa cells, to distinguish between distantly related HeLa and Polytomella cells, and between primary cultured rat neurons and glial cells. Finally we present a data integration workflow combining cryo-ET, light microscopy and MSI aligned in the same coordinate system to facilitate efficient data visualization and interpretation. We anticipate that this approach will change the way that structural biologists can approach cellular heterogeneity in cryo-ET, as cells can be identified and classified reliably post acquisition.

biophysics↗

bioRxiv2025

Neuronal processes contain the essential components for the late steps of ribosome biogenesis

Neurons rely on spatial and temporal control of protein synthesis to respond rapidly and locally to external stimuli, a process facilitated by the dynamic localization and modification of ribosomes. While previous research has shown that neuronal activity can regulate ribosome localization and modify translation rates, little is known about ribosomal assembly within neuronal processes. Here, we investigated the potential for local ribosome maturation in rat neurons using proteomics, RNA sequencing, and imaging methods. We detected an abundance of ribosome biogenesis factors (RBFs) in distal neuronal compartments, particularly those associated with the late stages of ribosome assembly. Moreover, we detected cytosolic pre-rRNA species in dendrites, alongside the enzymes necessary for their processing, suggesting that local ribosome maturation can occur far from the nucleus. These findings challenge conventional models that confine ribosome biogenesis to nuclear and perinuclear regions and suggest that neurons may fine-tune local protein synthesis by regulating ribosome assembly near synaptic sites. This mechanism may enable rapid modulation of the translational capacity in response to physiological changes, regulating synaptic plasticity and local protein synthesis in neurons. Significance StatementNeurons require precise spatial and temporal regulation of protein synthesis to adapt rapidly to external stimuli, particularly at synapses. Our study challenges the view that new ribosomes can be made exclusively near the nucleus and reveals that ribosome biogenesis factors and pre-rRNA processing enzymes are present in distal neuronal compartments. These findings suggest that ribosome maturation can occur locally in dendrites, enabling rapid, spatially targeted modulation of translational capacity. This mechanism provides a different framework for understanding how neurons regulate synaptic plasticity and adapt to physiological changes. By demonstrating that ribosome assembly may extend beyond the nucleus, this work highlights a previously unrecognized layer of neuronal protein synthesis control, potentially transforming our understanding of how neurons orchestrate local responses to environmental cues.

cell biology↗

bioRxiv2025

The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

Proteostasis - the maintenance of proteins at proper concentrations, conformations, and subcellular locations - is essential for cellular function and is governed by tightly regulated protein synthesis and degradation pathways. The Proteasome Maturation Protein (POMP) is a key chaperone involved in assembling the proteasome, the primary complex responsible for protein degradation. Despite the conserved role of POMP, its loss produces contrasting proteostatic effects in yeast and mammalian cells, pointing to additional, unexplored functions. In this study, we investigated the possibility that POMP plays a moonlighting role in proteostasis regulation. We discovered that upon proteasome disruption, POMP rapidly accumulates in the nucleolus in a manner dependent on HSF1 and reactive oxygen species (ROS). Proteomic analysis of POMP interactors revealed RNA processing factors and transcriptomic profiling showed that nucleolar POMP orchestrates a protective transcriptional program. Our findings indicate POMP is a built-in sensor and effector within the proteasome assembly pathway, capable of buffering disturbances in proteasome function through a novel, non-canonical role. Notably, this mechanism is developmentally controlled and active in neurodegenerative disease contexts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/650603v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@196cd2borg.highwire.dtl.DTLVardef@177d240org.highwire.dtl.DTLVardef@18c9bcborg.highwire.dtl.DTLVardef@483b94_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

An integrative model of AMPA receptor trafficking reveals the central contribution of local translation in subtype-specific kinetics

AMPA-type glutamate receptors (AMPARs) underlie most of the excitatory synaptic transmission in the brain and are crucial for implementing long-term synaptic plasticity. AMPARs are multi-protein complexes composed of two types of subunits: pore-forming subunits GluA1-4 that assemble in the endoplasmic reticulum and form the glutamate-gated ion channel, and auxiliary subunits that modulate receptor bio-physical properties and mediate their forward trafficking to the plasma membrane. Here, using a combination of theoretical and experimental approaches, we elucidate the kinetics of essential trafficking steps and the protein sources necessary to explain the experimentally observed distribution of AMPARs and the response of different AMPAR subtypes to LTP induction. Our data indicate that the mRNA coding for one of the most abundant AMPAR auxiliary subunits, CNIH-2, is abundant in dendrites. Consistent with this mRNA distribution, CNIH-2 is locally synthesized. In contrast, the pore-forming subunits GluA1 and GluA2 are mostly synthesized in the cell body. CNIH-2 synthesis increases after the (chemical) induction of long-term potentiation. Strikingly, the translation of CNIH-2 is required for the plasma membrane insertion of GluA2-containing receptors and not GluA1-homomeric AMPARs. Using the selective trafficking of GluA2-containing AMPARs by CNIH-2, our computational model can explain the distinct temporal profiles in response to plasticity induction of two major subtypes of AMPARs, the slow-response of the calciumimpermeable (GluA2-containing) and fast kinetics of the calcium-permeable (GluA2-lacking) AMPARs.

neuroscience↗

bioRxiv2024

Cell type-Specific In Vivo Proteomes with a Multi-copy Mutant Methionyl t-RNA Synthetase Mouse Line

The functional diversity of cells is driven by the different proteins they express. While improvements in protein labeling techniques have allowed for the measurement of proteomes with increased sensitivity, measuring cell type-specific proteomes in vivo remains challenging. One of the most useful pipelines is bioorthogonal non-canonical amino acid tagging (BONCAT) with the MetRS* system, consisting of a transgenic mouse line expressing a mutant methionyl-tRNA synthetase (MetRS*) controlled by Cre recombinase expression. This system allows for cell type-specific labeling of proteins with a non-canonical amino acid (azidonorleucine, ANL), which can be subsequently conjugated to affinity or fluorescent tags using click chemistry. Click-modified proteins can then be visualized, purified and identified. The reduction in sample complexity allows for the detection of small changes in protein composition. Here we describe a multicopy MetRS* mouse line (3xMetRS* mouse line), which exhibits markedly enhanced ANL protein labeling, boosting the sensitivity and temporal resolution of the system and eliminating the need for working under methionine depletion conditions. Cell type-specific in vivo labeling is possible even in heterozygous animals, thus offering an enormous advantage for crossing the line into mutation and disease-specific backgrounds. Using the 3xMetRS* line we identified the in vivo proteome of a sparse cell population - the dopaminergic neurons of the olfactory bulb and furthermore determined newly synthesized proteins after short labeling durations following a single intraperitoneal ANL injection.

cell biology↗

bioRxiv2024

The molecular diversity of hippocampal regions and strata at synaptic resolution revealed by integrated transcriptomic and proteomic profiling

The molecular diversity of neurons and their synapses underlies the different responses and plasticity profiles that drive all neural circuits and behavior. While the extent of this diversity has been partially revealed by transcriptomic and proteomic profiling, combined studies of neuronal transcripts and proteins are limited. Here, we used microdissection of mouse hippocampal subregions and CA1 strata and fluorescence-activated synaptosome sorting (FASS) to characterize the transcripts and proteins from different hippocampal neurons and their compartments with synaptic resolution. Parallel RNA-seq and LC-MS/MS of microdissections identified over 15,000 mRNA transcripts and 10,000 proteins, revealing thousands with local enrichment such as classes of glutamate receptors and voltage-gated potassium channels, myelin-associated molecules, and adhesion molecules. Synaptosome analysis further identified specific enrichment of molecules from collagen, ribosome, solute carrier, and receptor families at different synapses formed along CA1 neurons. By integrating mRNA and protein data, we defined clusters of co-regulated molecules such as adhesion and neurofilament proteins and transporter mRNAs, and found subsets of mRNA-protein pairs with strong correlation and anti-correlation in their abundance variation. Our findings comprise a rich resource on the molecular landscape of the hippocampus and its synapses that is accessible at syndive.org, and highlight the coordinated organization of transcripts and proteins between regions, neuronal compartments, and synapses.

neuroscience↗

bioRxiv2023

The proteomic landscape of synaptic diversity across brain regions and cell types

Brain function relies on communication via neuronal synapses. Neurons build and diversify synaptic contacts using different protein combinations that define the specificity, function and plasticity potential of synapses. More than a thousand proteins have been globally identified in both pre- and postsynaptic compartments, providing substantial potential for synaptic diversity. While there is ample evidence of diverse synaptic structures, states or functional properties, the diversity of the underlying individual synaptic proteomes remains largely unexplored. Here we used 7 different Cre-driver mouse lines crossed with a floxed mouse line in which the presynaptic terminals were fluorescently labeled (SypTOM) to identify the proteomes that underlie synaptic diversity. We combined microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting to isolate and analyze using quantitative mass spectrometry 18 types of synapses and their underlying synaptic proteomes. We discovered ~1800 unique synapse type-enriched proteins and allocated thousands of proteins to different types of synapses. We identify commonly shared synaptic protein modules and highlight the hotspots for proteome specialization. A protein-protein correlation network classifies proteins into modules and their association with synaptic traits reveals synaptic protein communities that correlate with either neurotransmitter glutamate or GABA. Finally, we reveal specializations and commonalities of the striatal dopaminergic proteome and outline the proteome diversity of synapses formed by parvalbumin, somatostatin and vasoactive intestinal peptide-expressing cortical interneuron subtypes, highlighting proteome signatures that relate to their functional properties. This study opens the door for molecular systems-biology analysis of synapses and provides a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.

neuroscience↗

bioRxiv2022

An Abundance of Free Proteasomal Regulatory (19S) Particles Regulate Neuronal Synapses Independent of the Proteasome

The major protein-degradation machine, the proteasome, functions at brain synapses and regulates long-term information storage. Here we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles that recognize and degrade substrates, are differentially distributed within individual rat cortical neurons. Using superresolution microscopy, we discovered a surprising abundance of free particles (19S) near synapses. The free neuronal 19S particles bind and deubiquitylate Lys63-ubiquitin, a non-proteasome targeting ub-linkage. Pull-down assays revealed a significant over-representation of synaptic molecules as Lys63 interactors. Inhibition of 19S deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses. One-Sentence SummaryThe 19S subcomplex of the major protein-degradation machine, the proteasome, functions without its 20S partner to populate and regulate synapses.

neuroscience↗

bioRxiv2021

Co-translational assembly counteracts promiscuous interactions

During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examined structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally tested candidate structural motifs and identified several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assembled co-translationally in only one but not all of the relevant assembly pathways. Our results highlight the regulatory complexity of assembly pathways.

molecular biology↗

bioRxiv2021

The novel Rab5 effector FERRY links early endosomes with the translation machinery

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here we show that the novel Rab5 effector Five-subunit Endosomal Rab5 and RNA/ribosome intermediarY, FERRY complex recruits mRNAs and ribosomes to early endosomes, through direct mRNA interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA and protein levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

cell biology↗

bioRxiv2021

Dynamic bi-directional phosphorylation events associated with the reciprocal regulation of synapses during homeostatic up- and down-scaling.

Homeostatic synaptic scaling allows for bi-directional adjustment of the strength of synaptic connections in response to changes in their input. Protein phosphorylation modulates many neuronal and synaptic processes, but it has not been studied on a global, proteome-wide scale during synaptic scaling. To examine this, we used LC-MS/MS analyses to measure changes in the phosphoproteome in response to up- or down-scaling in cultured cortical neurons over minutes to 24 hours. Out of 45,000 phosphorylation events measured, ~3,300 (associated with 1,280 phospho-proteins) were regulated by homeostatic scaling. The activity-sensitive phosphoproteins were predominantly located at synapses and involved in cytoskeletal reorganization. We identified many early transient phosphorylation events which could serve as sensors for the activity offset as well as late and/or persistent phosphoregulation that could represent effector mechanisms driving the homeostatic response. Much of the persistent phosphorylation was reciprocally regulated by up- or down-scaling, suggesting that the mechanisms underlying these two poles of synaptic regulation make use of a common signaling axis.

neuroscience↗

bioRxiv2021

Neuronal ribosomes dynamically exchange ribosomal proteins in a context-dependent manner.

Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs quickly associated with translating ribosomes in the cytoplasm and that this incorporation is independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (in response to oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing remote neuronal synapses a rapid solution to the relatively slow and energy-demanding requirement of nuclear ribosome biogenesis.

molecular biology↗

bioRxiv2020

Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons

The localization and translation of mRNAs to dendrites and axons maintains and modifies the local proteome of neurons, and is essential for synaptic plasticity. Although significant efforts have allowed the identification of localized mRNAs in excitatory neurons, it is still unclear whether interneurons also localize a large population of mRNAs. In addition, the variability in the population of localized mRNAs within and between cell-types is unknown. Here we developed a method for the transcriptomic characterization of a single neurons subcellular compartments, which combines laser capture microdissection with scRNA-seq. This allowed us to separately profile the dendritic and somatic transcriptomes of individual rat hippocampal neurons and investigate the relation in mRNA abundances between the soma and dendrites of single glutamatergic and GABAergic neurons. We identified two types of glutamatergic and three types of GABAergic interneurons and we found that, like their excitatory counterparts, interneurons contain a rich repertoire of ~4000 mRNAs. The individual somatic transcriptomes exhibited more cell type-specific features than their associated dendritic transcriptomes. The detection and abundance of dendritic mRNAs was not always simply predicted by their somatic counterparts. Finally, using cell-type specific metabolic labelling of isolated neurites, we demonstrated that the processes not only of Glutamatergic but also of GABAergic neurons are capable of local translation, suggesting mRNA localization and local translation is a general property of neurons.

neuroscience↗

bioRxiv2020

The mRNA translation landscape in the synaptic neuropil.

To form and modify synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes unique logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of mRNAs near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally1. It is not known, however if, how and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA-seq and Ribo-seq from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies as well as dendrites and axons (neuropil). More than 4800 transcripts were translated in synaptic regions. Thousands of transcripts were differentially translated between somatic and synaptic regions, with scaffold and signaling molecules mostly arising from local sources. Furthermore, specific mRNA features were identified that regulate the efficiency of mRNA translation. The findings overturn the view that local translation is a minor source of synaptic protein2 and indicate that on-site translational control is an important mechanism to control synaptic strength.

neuroscience↗