Biology subjects

Tushev, G.

Publications and source records attributed to Tushev, G..

12 recordsLinked to original sources

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

Ischemic injury triggers a protective microglial phenotype in models of Aβ pathology

Microglia are highly plastic cells that are capable of integrating subsequent insults. As the majority of Alzheimers Disease (AD) patients also show cerebrovascular pathology, we here aimed to dissect the interactions between AD and ischemic brain injury on the microglial response to amyloid beta (A{beta}) pathology. Surprisingly, we find that ischemic stroke in the presence of cerebral {beta}-amyloidosis results in the generation of a novel neuroprotective microglial phenotype. These microglia drive a rapid accumulation of highly dense A{beta} plaques that exhibit a relatively benign nature and are strikingly similar to A{beta} plaques observed in patients that are resilient to AD pathology. Thus, our data do not only highlight the impact of a co-morbid state of brain ischemia and A{beta} pathology on the microglial phenotype but also identify novel molecular pathways that may serve to promote beneficial microglial functions in AD.

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

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

Chromosome-scale genome assembly of the European common cuttlefish Sepia officinalis

Coleoid cephalopods, a subclass of mollusks that includes octopuses, cuttlefish and squid, exhibit sophisticated biological features such as dynamic and neurally driven camouflage behavior, inter-individual communication, single-lens camera-like eyes, the largest brains among invertebrates and a distinctive embryonic development. The common cuttlefish Sepia officinalis has served as a model organism in various research fields, spanning biophysics, neurobiology, behavior, evolution, ecology and biomechanics. More recently, it has become a model to investigate the neural mechanisms underlying cephalopod camouflage, using quantitative behavioral approaches alongside molecular techniques to characterize the identity, evolution and development of neuronal cell types. Despite significant interest in this animal, a high-quality, annotated genome of this species is still lacking. To address this, we sequenced and assembled a chromosome-scale genome for S. officinalis. Our assembly spans 5.68 billion base pairs and comprises 1n=47 repeat-rich chromosome scaffolds. This was unexpected because the haploid karyotypes of other decapods indicate 46 chromosomes. Detailed comparisons of our data to those from published decapod genome assemblies and to another recent genome assembly of S. officinalis (itself suggesting 1n=49 chromosomes) in fact revealed clear homologies between 46 scaffolds across all the datasets. The discrepancies between datasets are explained by highly repetitive regions, impairing proper read alignments. We conclude that the true karyotype of S. officinalis is probably 1n=46 chromosomes, a likely ancestral and if true, conserved decapod karyotype. Our results include a comprehensive gene annotation and full-length transcript prediction, which we used to characterize orthologous gene families across mollusks. We identified several large-scale expansions specific to cephalopods, with many genes specific to neural or non-neural tissues of adult S. officinalis. In summary, this genome should provide a valuable resource for future research on the evolution, brain organization, information processing, development and behavior in this important clade.

genomics↗

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↗

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↗

bioRxiv2019

Monosomes actively translate synaptic mRNAs in neuronal processes

In order to deal with their huge volume and complex morphology, neurons localize mRNAs and ribosomes near synapses to produce proteins locally. A relative paucity of polyribosomes (considered the active sites of translation) detected in electron micrographs of neuronal processes (axons and dendrites), however, has suggested a rather limited capacity for local protein synthesis. Polysome profiling together with ribosome footprinting of microdissected synaptic regions revealed that a surprisingly high number of dendritic and/or axonal transcripts were predominantly associated with monosomes (single ribosomes). Contrary to prevailing views, the neuronal monosomes were in the process of active protein synthesis (e.g. they exhibited elongation). Most mRNAs showed a similar translational status in both compartments, but some transcripts exhibited differential ribosome occupancy in the somata and neuropil. Strikingly, monosome-preferred transcripts often encoded high-abundance synaptic proteins. This work suggests a significant contribution of monosome translation to the maintenance of the local neuronal proteome. This mode of translation can presumably solve some of restricted space issues (given the large size of polysomes) and also increase the diversity of proteins made from a limited number of ribosomes available in dendrites and axons.

neuroscience↗

bioRxiv2017

Poly(A) Binding Protein Nuclear 1 regulates the polyadenylation of key synaptic plasticity genes and plays a role in homeostatic plasticity

Polyadenylation is a nuclear process that involves the endonucleolytic cleavage of RNA transcripts and the addition of poly(A) tails. The cleavage often takes place at different positions within the same RNA transcript, generating alternative 3ends. Polyadenylation regulates mRNA localization, stability and translation and is likely to regulate complex processes such as synapse formation, synaptic plasticity and memory. Here we examined whether PolyA Binding Protein Nuclear 1 (Pabpn1), an RNA binding protein known to regulate alternative polyadenylation and polyA tail length in other systems, regulates neuronal mRNA function. Using immunocytochemistry we determined that Pabpn1 is present in both hippocampal slices and cultured hippocampal neurons. Applying shRNAs to knock-down Pabpn1 we discovered that Pabpn1 regulates the mRNA abundance and localization of key synaptic plasticity genes including Calcium/Calmodulin Dependent Protein Kinase II Alpha (Camk2a) and Glutamate Ionotropic Receptor AMPA Type Subunit 2 (Gria2). Furthermore, Pabpn1 knock-down prevented the homeostatic scaling of synaptic transmission elicited by bicuculline. These data demonstrate a link between Pabpn1, polyadenylation and neuronal plasticity.

neuroscience↗