Biology subjects

Altelaar, M.

Publications and source records attributed to Altelaar, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatiotemporal proteomics elucidates trafficking and localization of Neuroligins to the axon initial segment plasma membrane

Neuronal development and function rely on precise sorting of membrane proteins to distinct neuronal domains, yet the underlying mechanisms remain incompletely understood. Here, we investigated the trafficking of two highly homologous NLGNs (NLGN1 and 2) which distinctly localize and function at excitatory and inhibitory synapses. By using spatiotemporal proteomics, genetic engineering and microscopy in CNS cultured neurons and organotypic slices, we dissected the itinerary of biosynthetic NLGNs, identifying co-cargoes and sorting routes to dendrites and the AIS. At the AIS, which contains only inhibitory synapses, both NLGN1 and NLGN2 are locally exocytosed. While NLGN2 is stably recruited to axo-axonic synapses by its extracellular domain and modulated by activity, NLGN1 is retrieved by endocytic mechanisms. We propose that biosynthetic NLGNs are co-sorted to the AIS PM where they detect pre-synaptic type. These findings may help to elucidate the role of AIS-localized NLGNs in shaping AIS structural and functional plasticity. HighlightsO_LISpatiotemporal proteomics elucidates interactome of biosynthetic NLGN1 in neurons C_LIO_LINLGN1 and NLGN2 are targeted to the AIS by kinesin-1 C_LIO_LINLGN2, but not NLGN1, is retained at axo-axonic synapses and modulated by activity C_LIO_LINLGN stability at the AIS plasma membrane is determined by the ectodomain C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/733123v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@a45909org.highwire.dtl.DTLVardef@1d171b0org.highwire.dtl.DTLVardef@17f132aorg.highwire.dtl.DTLVardef@1c33a8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Stoichiometry-dependent specificity in biotin enrichment: a benchmarking framework for proximity labeling proteomics

Proximity labeling methods (including, BioID, TurboID, ultraID), along with surface proteomics and microdomain mapping, enable proteome-wide identification of spatially proximal proteins via MS-based analysis. These workflows require specific enrichment of biotinylated proteins using affinity purification, yet enrichment specificity can often be compromised by non-specifically bound proteins. As labeling strategies are increasingly applied to complex biological samples with low protein input or low biotin stoichiometry, accurately distinguishing true targets from background becomes a major analytical challenge. Despite its critical impact on data quality and interpretation, the influence of biotinylation level and protein input on enrichment performance remains poorly characterized, limiting the reliability of proximity labeling experiments. To address this, we establish a quantitative benchmarking framework that systematically evaluates biotin enrichment under controlled conditions, including scenarios of low biotin stoichiometry. Using this setup, we show that enrichment specificity strongly depends on biotin stoichiometry: higher levels of biotinylation in samples yield high specificity, whereas low biotinylation increases non-specific background. Reduced protein input further limits recovery of true targets, yet maintains enrichment specificity, highlighting sensitivity constraints of enrichment-based workflows. We apply this framework to biotinylated extracellular vesicle (EV) cargo uptake in recipient cells using ultraID-CD63 labeling. Detection of the most abundant EV cargo proteins under low biotinylation conditions indicates that current workflows approach the lower bounds of biotin enrichment sensitivity. Together, these standards provide a practical reference for evaluating and optimizing biotin enrichment workflows, supporting quantitative and reproducible proximity labeling in proteomics.

molecular biology↗

bioRxiv2026

The phosphoS655 Alzheimer's Amyloid Precursor Protein (APP) interactome in neuronal differentiation

The Alzheimers Amyloid Precursor Protein (APP) has determinant roles in neuronal development and function, both in its full-length conformation and as some of its proteolytic peptides, particularly secreted (s)APPa. Given that APP phosphorylation tightly regulates its trafficking, proteolysis, and protein-protein binding, it consequently affects several APP functions. The S655 residue, located in the basolateral sorting motif YTSI at APP C-terminus has been observed to be phosphorylated in mature full-length APP and its C-terminal fragments. Previously observed to modify APPs protein interactions, resulting in altered endolysosomal trafficking, andincreased half-life and sAPPa generation, phosphoS655 APP has potential to modulate APP-mediated neuronal differentiation. To study the phosphoS655 differential interactome relevant for neuronal differentiation, SH-SY5Y cells expressing Wt or S655 phosphomutants APP-GFP were differentiated at two time points. APP-GFP and their respective interacting partners were immunoprecipitated using GFP-trap, and interactors identified by mass spectrometry. Both dephospho and phosphoS655 interactomes were generally enriched in similar processes, primarily RNA processing and translation, as well as signal transduction, metabolism, and cytoskeleton remodeling. The smaller phosphoS655 interactome contributes for functional specialization via binding to e.g. FUBP3, ELAVL4, ATXN2, Tubulin, INA. Several of these specific binding partners are known to promote neurite outgrowth and likely underlie our experimental observation that phosphoS655 APP promotes neuritogenesis, particularly the formation of longer neuritic extensions. These results are not only important for the body of knowledge on this Alzheimers disease core protein, but may also aid in future therapies against this disease.

neuroscience↗

bioRxiv2025

GDF15 regulates necroptotic cell death through direct interaction with RIPK3

Enhancing the immunogenicity of tumor cells is a major objective in cancer therapy, particularly for tumors with low immune cell infiltration scores. Inducing immunogenic forms of programmed cell death (PCD) offers a promising strategy to strengthen anti-tumor immunity and improve therapeutic outcomes. Necroptosis, a highly inflammatory form of regulated cell death triggered by TNF signaling, can elicit robust immune activation. However, its regulation in tumor cells remains incompletely understood, limiting its therapeutic exploitation. To investigate the protein-protein interactions that govern necroptotic cell death in tumor cells, we established a co-Immunoprecipitation - Mass Spectrometry (coIP-MS) workflow using RIPK3, the central effector kinase driving necroptosis in TNF-induced signaling, as bait. This unbiased proteomic approach enables the identification of candidate regulators directly associated with the necrosome complex components under active necroptotic conditions. Among identified candidates, TCOF1 and GDF15 emerged as previously unrecognized modulators, with functional knockout of either gene markedly enhancing necroptotic cell death in tumor cells. Reciprocal IP experiments confirmed a direct interaction between GDF15 and RIPK3, supporting its mechanistic role as a negative regulator that suppresses necroptotic signaling. Thus, our findings extend the function of GDF15 beyond its established role in inflammation, uncovering an additional layer of regulation at the level of cell-intrinsic death signaling. Collectively, our findings position GDF15 as a RIPK3-interacting "brake" on necroptotic cell death and highlight TCOF1 as an additional inhibitory node. Our study underscores the potential of targeting necroptosis-suppressive mechanisms to influence PCD outcomes in tumors and demonstrates the power of coIP-MS for mapping TNF-induced interactions to reveal actionable molecular targets for tumor sensitization.

cell biology↗

bioRxiv2025

Decoding interaction-induced proteome changes in co-cultures with hybrid quantification and SILAC-directed real-time search

Intercellular communication between T cells and cancer cells plays a pivotal role in determining cancer cell survival or death. Yet, our understanding of this interaction remains incomplete. Methods to study heterotypic cell interactions are either limited to targeted studies relying on predefined set of proteins, or require cell separation, thus disrupting the native environment. Stable isotope labeling by amino acids in cell culture (SILAC) enables proteome distinction in heterologous co-cultures without the need for physical separation. But dynamic studies remain constrained by the need for numerous mass spectrometry (MS) runs, the challenges in detecting low-abundant proteins, particularly in immune cells and the limited data completeness due to the use of data-dependent MS1-based precursor quantification. To overcome these limitations, we evaluate the integration of SILAC with tandem mass tag (TMT) multiplexing and SILAC-directed real-time search (RTS). TMT labeling enables simultaneous analysis of multiple samples, while RTS-MS3 acquisition using SILAC-induced mass shifts as fixed modifications triggers MS3 scans for specific proteome populations within a mixed cell system, improving quantitative accuracy and proteome coverage for target protein populations. We benchmarked our acquisition methods using SILAC-labeled samples mixed at defined ratios and validated the approach in biologically relevant co-culture experiments. Additionally, we introduced a carrier channel to enhance detection of lower-abundant T cell proteins, while maintaining acceptable quantitative precision. Our results demonstrate that the combined SILAC-TMT-RTS strategy dramatically improves proteome depth, temporal resolution, and cell-type specificity for short-term co-culture interaction proteomics studies. In co-culture samples of T cells with non-small cell lung cancer cell lines that were either sensitive or resistant to T cell killing, our method revealed candidate mechanisms underlying their differential sensitivity. Our integrated approach combining SILAC, TMT and RTS to resolve cell-specific proteome dynamics in co-culture represents a novel and powerful advance.

cancer biology↗

bioRxiv2025

The axonal ER couples translation and secretion machineries for local delivery of axonal transmembrane proteins to promote axonal development

Neuronal development and function rely on the polarized sorting of transmembrane proteins (TMPs). Most TMPs follow a conventional secretory pathway confined to the cell soma, where ER-translated TMPs exit the ER through ERES to reach the Golgi, prior to PM delivery. We recently found that the axonal ER regulates local translation; however, how this is linked to secretion remains unknown. Here, we find that axonally translated TMPs exit the ER and reach the axonal PM via an ERES-dependent/Golgi-independent route. Intriguingly, we uncover a feedback loop linking translation and secretion at axonal ERES. HDLBP-dependent TMP synthesis regulates ERES formation, and axonal ERES in turn modulate TMP translation. Subsequently, the NRZ-SEC22B tethering complex links local TMP ER exit to PM delivery, dependent on ER-PM contacts. Axonal ERES and these associated molecular players support axon growth and bouton assembly. We propose a novel mechanism in which coupling of axonal TMP synthesis and secretion is essential for neuronal development.

cell biology↗

bioRxiv2025

Inhibition of TBK1/IKKe mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing

Resistance to immune cell-mediated cytotoxicity poses a significant challenge in cancer therapy, compromising the efficacy of immunotherapeutic approaches such as immune checkpoint blockade (ICB) treatment. To enhance therapy outcomes, it is crucial to identify interventions that can synergize with ICB therapy to overcome tumor resistance. Therefore, we need to define the cellular mechanisms that sensitize tumors to cytotoxic T cells. CD8 T cells rely on cytokines such as TNF to carry out their cytotoxicity against tumors, and recent findings link select tumor mutations in the TNF pathway to increased T cell killing, in a manner dependent on RIPK1 kinase. Here, we demonstrate that sensitized tumor cells fail to initiate inhibitory RIPK1 phosphorylation at site S25 upon T cell attack, thereby foregoing a pro-survival checkpoint early in TNF signal transduction. Consequently, tumor cells experiencing a loss of TNF-induced RIPK1 S25 phosphorylation exhibit increased RIPK1 activation and fail to recruit non-canonical IKK kinases (TBK1 and IKKe) to the TNFR1 complex. Functional knockouts of TBK1 and IKKe in melanoma cells result in heightened sensitivity not only in CD8 T cell but also in Natural Killer cell attacks. Our findings indicate that preventing TBK1 and IKKe recruitment to the TNF signaling complex, thereby blocking RIPK1 pro-survival phosphorylation and promoting direct RIPK1 activation, is a tractable strategy to increase tumor sensitivity to immune cell killing and has the potential to benefit current immunotherapy interventions.

cell biology↗

bioRxiv2024

ARID1A-induced transcriptional reprogramming rewires signalling responses to drug treatment in melanoma

Resistance to BRAF and MAPK inhibitors is a significant challenge in melanoma treatment, driven by adaptive and acquired mechanisms that allow tumour cells to evade therapy. Here, we examined early signalling responses to single and combined BRAF and MAPK inhibition in a BRAFV600E, drug-sensitive melanoma cell line and a drug-resistant ARID1A-knockout (KO) derivative. ARID1A, frequently mutated in melanoma, is associated with resistance and immune evasion. Using an innovative systems biology approach that integrates transcriptomics, proteomics, phosphoproteomics, and functional kinomics through matrix factorization and network analysis, we identified key signalling alterations and resistance mechanisms. We found that ARID1A-KO cells exhibited transcriptional rewiring, sustaining MAPK1/3 and JNK activity post-treatment, bypassing feedback sensitivity observed in parental cells. This rewiring suppressed PRKD1 activation, increased JUN activity--a central resistance network node--and disrupted PKC dynamics through elevated basal RTKs (e.g., EGFR, ROS1) and Ephrin receptor activity post-treatment. ARID1A mutations also reduced HLA-related protein expression and enriched extracellular matrix components, potentially limiting immune infiltration and reducing immunotherapy efficacy. Our graph-theoretical multi-omics approach uncovered novel resistance-associated signalling pathways, identifying PRKD1, JUN, and NCK1 as critical nodes. While receptor activation redundancies complicate single-target therapies, they also present opportunities for combination strategies. This study highlights ARID1As role in reshaping signalling and immune interactions, offering new insights into melanoma resistance mechanisms. By identifying actionable targets, including JUN and immune pathways, we provide a foundation for developing integrated therapeutic strategies to overcome resistance in BRAF/MAPK inhibitor-treated melanoma. One sentence summaryThis study reveals how ARID1A-mediated transcriptional rewiring drives resistance to MAPK inhibitors in melanoma by altering signalling pathways, immune interactions, and receptor dynamics, highlighting potential targets for combinatorial therapies.

systems biology↗

bioRxiv2024

Sensitivity to Vγ9Vδ2TCR T cells is imprinted after single mutations during early oncogenesis

V{gamma}9V{delta}2T cells have the unique ability to recognize a broad range of malignant transformed cells. The tumor targeting event involving BTN2A1 and BTN3A1 dimers on the tumor cell surface is critical, leading to full activation of the TCR. Although the molecular mechanisms governing TCR engagement and T cell activation are well-characterized, the role of V{gamma}9V{delta}2 T cells in cancer immune surveillance remains to be fully elucidated, particularly the mechanisms that enable these cells to discriminate between healthy and malignant cells at an early stage of malignant transformation. We employed two independent, genetically engineered step-wise mutagenesis models of human colorectal and breast cancer that mimic the transformation steps leading to tumor formation. We demonstrate that various single oncogenic mutations introduced into healthy organoids or cells, are sufficient to upregulate surface expressed BTN2A1 and enable V{gamma}9V{delta}2 TCR binding to tumor cells. However, full activation of T cells through a V{gamma}9V{delta}2TCR required additional subsequent phosphorylation of juxtamembrane (JTM) amino acids of BTN3A1, leading to the activating heterodimerization of BTN2A1 and 3A1. Using a protein interactome mapping pipeline, we identified PHLDB2, SYNJ2 and CARMIL1 as key players in controlling these delicate dual surface dynamics of BTN2A1 and 3A1 during early transformation. This mode of action allowed V{gamma}9V{delta}2TCR T cells to control tumors in vitro and in vivo, emphasizing the crucial role of these molecules from early mutagenesis, to advanced cancer stages, and highlighting the therapeutic potential of a V{gamma}9V{delta}2TCR.

cancer biology↗

bioRxiv2024

Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

Lysosomes are membrane-bound organelles critical for maintaining cellular homeostasis. Delivery of biosynthetic lysosomal proteins to lysosomes is crucial to orchestrate proper lysosomal function. However, it remains unknown how the delivery of biosynthetic lysosomal proteins to lysosomes is ensured in neurons, which are highly polarized cells. Here, we developed Protein Origin, Trafficking And Targeting to Organelle Mapping (POTATOMap), by combining trafficking synchronization and proximity-labelling based proteomics, to unravel the trafficking routes and interactome of the biosynthetic lysosomal membrane protein LAMP1 at specified time points. This approach, combined with advanced microscopy, enabled us to identify the neuronal domain-specific trafficking machineries of biosynthetic LAMP1. We revealed a role in replenishing axonal lysosomes, in delivery of newly synthesized axonal synaptic proteins, and interactions with RNA granules to facilitate hitchhiking in the axon. POTATOMap offers a robust approach to map out dynamic biosynthetic protein trafficking and interactome from their origin to destination.

cell biology↗

bioRxiv2024

NAC-mediated ribosome localization regulates cell fate and metabolism in intestinal stem cells

Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity, however it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we show that RNA translation is at the root of this interplay. We identify the nascent polypeptide-associated complex (NAC) as a key mediator of this process, and show that it regulates ISC metabolism by relocalizing ribosomes to the mitochondria. Upon NAC inhibition, intestinal cells show decreased import of mitochondrial proteins, which are needed for oxidative phosphorylation, and, consequently, enable the cell to maintain a stem cell identity. Furthermore, we show that overexpression of NAC is sufficient to drive mitochondrial respiration and promote ISC identity. Ultimately, our results reveal the pivotal role of ribosome localization in regulating mitochondrial metabolism and ISC function. TeaserThe location of ribosomes in cells is regulated, and defines the fate of intestinal stem cells.

molecular biology↗

bioRxiv2023

An "alert state" ribosome population acts as a master regulator of cytokine mediated processes

Inflammatory cytokines are pivotal to immune responses. Upon cytokine exposure, cells enter an "alert-state" that enhances their visibility to the immune system. Here, we identified an "alert-state" subpopulation of ribosomes (ASRs) defined by the presence of the P-stalk. We show that ASRs are formed in response to cytokines linked to tumor immunity, and are involved in the preferential translation of mRNAs vital for the cytokine response. Mechanistically, ASRs are required for the efficient translation of transmembrane domains of receptor molecules involved in cytokine-mediated processes. Importantly, loss of the ASR prevents CD8+ T cell recognition and killing, and inhibitory cytokines like TGF{beta} hinder ASR formation, suggesting that the ASR is a central regulatory hub upon which multiple signals converge. Thus, the ASR is an essential mediator of the cellular rewiring that occurs following cytokine exposure, via the translational regulation of this process.

immunology↗

bioRxiv2023

A genome-wide CRISPR screen in human prostate cancer cells reveals drivers of macrophage-mediated cell killing and positions AR as a tumor-intrinsic immunomodulator

The crosstalk between prostate cancer (PCa) cells and the tumor microenvironment plays a pivotal role in disease progression and metastasis and could provide novel opportunities for patient treatment. Macrophages are the most abundant immune cells in the prostate tumor microenvironment (TME) and are capable of killing tumor cells. To identify genes in the tumor cells that are critical for macrophage-mediated killing, we performed a genome-wide co-culture CRISPR screen and identified AR, PRKCD, and multiple components of the NF-{kappa}B pathway as hits, whose expression in the tumor cell are essential for being targeted and killed by macrophages. These data position AR signaling as an immunomodulator, and confirmed by androgen-deprivation experiments, that rendered hormone-deprived tumor cells resistant to macrophage-mediated killing. Proteomic analyses showed a downregulation of oxidative phosphorylation in the PRKCD- and IKBKG-KO cells compared to the control, suggesting impaired mitochondrial function, which was confirmed by electron microscopy analyses. Furthermore, phosphoproteomic analyses revealed that all hits impaired ferroptosis signaling, which was validated transcriptionally using samples from a neoadjuvant clinical trial with the AR-inhibitor enzalutamide. Collectively, our data demonstrate that AR functions together with the PRKCD and the NF-{kappa}B pathway to evade macrophage-mediated killing. As hormonal intervention represents the mainstay therapy for treatment of prostate cancer patients, our findings may have direct implications and provide a plausible explanation for the clinically observed persistence of tumor cells despite androgen deprivation therapy.

cancer biology↗

bioRxiv2023

Elucidating Fibroblast Growth Factor-induced kinome dynamics using targeted mass spectrometry and dynamic modeling

Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that, activated by their ligands, elicit a wide range of health and disease-related processes, such as cell proliferation and the epithelial-to-mesenchymal transition (EMT). The detailed molecular pathway dynamics that coordinate these responses have remained to be determined. To elucidate these, we stimulated MCF-7 breast cancer cells with either FGF2, FGF3, FGF4, FGF10, or FGF19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phospho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals tight regulation of the RAF kinase family.

systems biology↗

bioRxiv2022

PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription

How steroid hormone receptors (SHRs) orchestrate transcriptional activity remains only partly understood. Upon activation, SHRs bind the genome and recruit their co-regulators, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we comprehensively dissected the Glucocorticoid Receptor (GR) co-regulatory complex involved in gene-target regulation. We describe a novel functional cross-talk between PAXIP1 and the cohesin subunit STAG2 that is critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on the genome, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. Moreover, in lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.

genomics↗

bioRxiv2022

Axonal ER tubules regulate local translation via P180/RRBP1-mediated ribosome interactions

Local mRNA translation in axons is critical for the spatial and temporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons, however how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with ER tubules in the axon in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation in an mRNA-dependent manner. Our results establish an important role for the axonal ER in localizing mRNA translation and in dynamically regulating the axonal proteome in response to neuronal stimuli.

neuroscience↗

bioRxiv2022

Predicting treatment outcome using kinome activity profiling in HER2+ breast cancer biopsies

In this study, we measured the kinase activity profiles of 32 pre-treatment tumour biopsies of HER2-positive breast cancer patients. The aim of this study was to assess the prognostic potential of kinase activity levels to identify potential mechanisms of resistance and to predict treatment success of HER2-targeted therapy combined with chemotherapy. Indeed, our system-wide kinase activity analysis, based on targeted mass spectrometry measurement of kinase activation loops, allowed us to link kinase activity to treatment response. Overall, high kinase activity in the HER2-pathway was associated with good treatment outcome. Furthermore, we found eleven kinases differentially regulated between treatment outcome groups. Amongst those, well-known players in therapy resistance were found, such as p38a, ERK and FAK, as well as a potential new player in drug resistance, namely MARK. Lastly, we defined an optimal signature of four kinases in a multiple logistic regression diagnostic test for prediction of treatment outcome (AUC=0.926). This kinase signature showed high sensitivity and specificity, indicating its potential as predictive biomarker for treatment success of HER2-targeted therapy.

cancer biology↗

bioRxiv2022

Amyotrophic Lateral Sclerosis Proteomic Signature And Treatment With Mesenchymal Stem Cell-derived Extracellular Vesicles

Motor neurons (MNs) derived from human induced pluripotent stem cells (iPSCs) offer a powerful model to study motor neuron diseases, such as amyotrophic lateral sclerosis (ALS). While widely used, our knowledge of the proteomic changes in these models is rather rudimentary. In this study, we conducted a comparative proteomic analysis of iPSC-derived MNs carrying ALS-associated mutations in C9ORF72, TARDBP, or FUS. This revealed both mutation-specific and shared proteomic signatures, unveiling common and divergent disease mechanisms. Using these new insights, we then evaluated the therapeutic potential of extracellular vesicles from mesenchymal stromal/stem cells (MSC-EVs). These experiments showed a functional effect of MSC-EVs in ALS-FUS MNs in vitro and their ability to reverse proteomic changes more generally in MNs with different ALS genetic backgrounds. These findings highlight key molecular pathways involved in ALS at the protein level and support the potential of MSC-EVs as a versatile therapeutic approach. Highlights- This study determines ALS-associated proteomic signatures in iPSC motor neurons (MNs) - Proteomic signatures indicate common and divergent disease mechanisms in MNs - MSC-EV treatment restores neurite growth defects in FUS-ALS MNs - MSC-EVs restore proteomic changes in MNs carrying different ALS mutations

neuroscience↗