Biology subjects

Garrido, A.

Publications and source records attributed to Garrido, A..

8 recordsLinked to original sources

bioRxiv2026

Capturing Cardiomyocyte Cell-to-Cell Heterogeneity via Shotgun Single Cell Top-Down Proteomics

Individual cells exhibit distinct molecular landscapes shaped by proteins and the diverse functional repertoire of their corresponding proteoforms. These structurally diverse variants (e.g., post-translationally modified including truncated proteolyzed forms) collectively orchestrate cellular functions. However, resolving proteoform heterogeneity at single-cell (SC) resolution remains a significant analytical challenge. Here, we present a shotgun SC top-down proteomics (SC-TDP) strategy that enables direct, unbiased proteoform profiling from single cardiomyocytes. Across 13 individual cardiomyocytes isolated from mouse heart, we identified a total of 57 proteins represented by 165 distinct proteoforms, including phosphorylated, succinylated, trimethylated, truncated, amongst others. Notably, proteoform composition varied substantially among cells, revealing a previously unrecognized level of molecular heterogeneity among cardiomyocytes. Together, these findings establish SC-TDP as a powerful tool for uncovering the proteoform diversity at the SC level. Our strategy paves the way for defining functional heterogeneity in cardiac tissue with unprecedented molecular resolution, enabling direct examination of the proteoform landscape that underlies cellular identity and physiology.

cell biology↗

bioRxiv2025

Phospho-signalling deregulation of the mTOR pathway in fibroblasts from LRRK2 Parkinson s patients

Activating mutations at the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent genetic cause of Parkinsons Disease (PD). Using in-depth mass-spectrometry, here we analysed the impact of LRRK2 mutations on the proteome and phospho-proteome of skin fibroblasts from a large LRRK2 clinical cohort from Spain. The cohort encompassed G2019S LRRK2-associated PD (L2PD) (n=15), G2019S LRRK2 non-manifesting carriers (L2NMCs) (n=13), idiopathic PD (iPD) (n=12), and control subjects (n=14) (total n=54). The proteome analysis revealed 89 differential proteins in G2019S L2PD (58 down/ 31 up) and 168 in G2019S L2NMCs (127 down / 41 up) compared to controls. Most of these proteome changes involved deficits in proteins related to mitochondrial energy and ribosomal protein synthesis. At the phospho-proteome level, we identified 394 differential phospho-sites in G2019S L2PD (188 hyper / 206 hypo), 367 in G2019S L2NMCs (215 hyper / 152 hypo), and 428 in iPD (214 hyper / 214 hypo) compared with controls. These phospho-proteomic changes included hyper-phosphorylated proteins associated with the mTOR and Rho GTPase pathways. Notably, the top hyper-phosphorylated hit in G2019S carriers was pThr37/46 4E-BP1, a key effector of the mTOR pathway controlling protein synthesis, and this protein was also deregulated in iPD. In summary, our study uncovers peripheral deregulation of mTOR phospho-signalling associated with LRRK2 mutations, which is related to deficits in mitochondrial and ribosomal proteins, and affects converging pathways altered in iPD.

neuroscience↗

bioRxiv2025

Polycomb repression works without Siesta

Polycomb group proteins mediate epigenetic repression via multi-subunit complexes, including canonical Polycomb Repressive Complex 1 (PRC1), which monoubiquitylates histone H2A and binds histone H3 tri-methylated at lysine 27 (H3K27me3). The RING1 subunit of PRC1, critical for H2A ubiquitylation, forms other complexes. These variant RING1 complexes also ubiquitylate H2A but cannot bind H3K27me3, and their evolutionary origins and functions are debated. Using Drosophila genetics, we discovered that canonical PRC1 and variant RING1 complexes ubiquitylate H2A at distinct genomic regions. We established that the sole Drosophila PCGF protein specific for variant RING1 complexes, which we named Siesta, is not required for epigenetic repression of developmental genes, but controls larval locomotion independently of H2A ubiquitylation. Leveraging a massively parallel transgenic approach, we demonstrated that H2A ubiquitylation has minimal impact on transcriptional repression. Our findings imply that variant RING1 complexes operate outside the Polycomb regulatory system and that the popular PRC1 nomenclature needs revision.

genetics↗

bioRxiv2024

Comparison of Fixed Single Cell RNA-seq Methods to Enable Transcriptome Profiling of Neutrophils in Clinical Samples

Monitoring neutrophil gene expression is a powerful tool for understanding disease mechanisms, developing new diagnostics, therapies and optimizing clinical trials. Neutrophils are sensitive to the processing, storage and transportation steps that are involved in clinical sample analysis. This study is the first to evaluate the capabilities of technologies from 10X Genomics, PARSE Biosciences, and HIVE (Honeycomb Biotechnologies) to generate high-quality RNA data from human blood-derived neutrophils. Our comparative analysis shows that all methods produced high quality data, importantly capturing the transcriptomes of neutrophils. 10X FLEX cell populations in particular showed a close concordance with the flow cytometry data. Here, we establish a reliable single-cell RNA sequencing workflow for neutrophils in clinical trials: we offer guidelines on sample collection to preserve RNA quality and demonstrate how each method performs in capturing sensitive cell populations in clinical practice.

genomics↗

bioRxiv2024

Cytotoxic CD4+ T cells in the bone marrow compromise healthy ageing by enhancing granulopoiesis

Neutrophils are the most abundant leukocytes in the blood, with numbers further increasing with age. Despite their essential role as a primary line of defense, neutrophils can contribute to tissue damage and age-related diseases 1 and a high neutrophil-to-lymphocyte ratio predicts all causes of mortality in the elderly 2-5. However, the precise mechanisms driving enhanced neutrophil generation during ageing remain poorly understood. Here, we show that a subset of CD4+ T cells with a cytotoxic phenotype (CD4+ CTLs) producing the chemokine CCL5 and harbouring dysfunctional mitochondria, infiltrate the bone marrow and induce granulopoiesis in aged mice. During ageing, hematopoietic stem cells upregulate CCR5, the primary receptor for CCL5, and its deficiency limits the T cell-mediated induction of granulopoiesis and neutrophil output. Treatment with the FDA-approved CCR5 inhibitor Maraviroc decreases granulopoiesis and lowers the levels of circulatory and tissue-infiltrating neutrophils, ameliorating multiple ageing biomarkers and improving functional outcomes in aged mice. These findings suggest that age-associated alterations in T cells reduce health outcomes by remodelling the bone marrow niche and enhancing neutrophil generation. Consequently, interventions to disrupt the interplay between T cells and hematopoietic stem cells hold substantial therapeutic potential to ameliorate age-associated diseases.

immunology↗

bioRxiv2022

The YAP-TEAD complex promotes senescent cell survival by lowering endoplasmic reticulum stress

Sublethal cell damage can trigger a complex adaptive program known as senescence, characterized by growth arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP). As senescent cells accumulating in aging organs are linked to many age-associated diseases, senotherapeutic strategies are actively sought to eliminate them. Here, a whole-genome CRISPR knockout screen revealed that proteins in the YAP-TEAD pathway influenced senescent cell viability. Accordingly, treating senescent cells with a drug that inhibited this pathway, Verteporfin (VPF), selectively triggered apoptotic cell death and derepressed DDIT4, in turn inhibiting mTOR. Reducing mTOR function in senescent cells diminished endoplasmic reticulum (ER) biogenesis, causing ER stress and apoptosis due to high demands on ER function by the SASP. Importantly, VPF treatment decreased senescent cell numbers in the organs of old mice and mice exhibiting doxorubicin-induced senescence. We present a novel senolytic strategy that eliminates senescent cells by hindering ER activity required for SASP production.

cell biology↗

bioRxiv2022

Immunosuppressive tocilizumab prevents astrocyte induced neurotoxicity in hiPSC-LRRK2 Parkinson's disease by targeting receptor interleukin-6

Parkinsons disease (PD) is associated with premature death of dopamine-producing neurons in the brain. Previous studies have shown that astrocytes of PD patients may contribute to neuronal degeneration by mechanisms involving both direct cell-to-cell contact and transfer of soluble molecules. Since it has been proposed that PD patients exhibit an overall pro-inflammatory state, and since astrocytes are key mediators of the inflammation response in the brain, here we sought to address whether astrocyte-mediated inflammatory signaling could contribute to PD neuropathology. For this purpose, we generated astrocytes from induced pluripotent stem cells (iPSCs) representing PD patients and healthy controls. Transcriptomic analyses identified a unique inflammatory gene expression signature in PD astrocytes compared to controls. In particular, the pro-inflammatory cytokine IL-6 was found to be highly expressed and released by PD astrocytes, and to induce toxicity in dopamine neurons. Mechanistically, neuronal cell death was mediated by IL-6 signaling via IL-6 receptor (IL-6R) expressed in human PD neurons, leading to downstream activation of STAT3. Importantly, astrocyte-induced cell death in PD disease midbrain neurons could be prevented by blocking IL6R-mediated signaling using clinically available antibodies. Moreover, examination of postmortem tissue brain of early-stage PD patients uncovered increased numbers of dopamine neurons overexpressing IL-6R and of reactive astrocytes overexpressing IL-6, compared to healthy brains. Our findings highlight the potential role of astrocyte-mediated inflammatory signaling in neuronal loss in PD, and open the way for new therapies based on IL-6 immunomodulation for preventing PD pathogenesis.

neuroscience↗

bioRxiv2022

Fanca and p53 genes cooperate to suppress oral SCC in transgenic mice

Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3-4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research of new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with a high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA.

cancer biology↗