Biology subjects

Lafarga, V.

Publications and source records attributed to Lafarga, V..

9 recordsLinked to original sources

bioRxiv2026

Modulation of retroviral capsid assembly halts ARC-mediated TDP-43 intercellular spreading

Intercellular propagation of pathological TDP-43 drives the progression of ALS and FTD, yet the mechanisms enabling transmission remain elusive. Here, we demonstrate that Activity-regulated cytoskeleton associated protein (ARC/Arg3.1) is pathologically subverted to act as a retroviral-like capsid vehicle for TDP-43 spreading. In a Drosophila model, we reveal that massive Arc1 upregulation drives glia-to-neuron TDP-43 seeding, while its genetic ablation halts pathological transfer, improves motor function, and extends survival. In parallel human cellular models, stress-induced ARC colocalizes with TDP-43 to orchestrate its intercellular transmission. Guided by ARCs capsid architecture, we reproposed Lenacapavir, an FDA-approved HIV-1 capsid modulator, as a stable binder of ARC/Arg3.1 capsid interfaces. Lenacapavir treatment effectively blocks TDP-43 propagation in vitro and rescues disease phenotypes in vivo. Our findings establish ARC as a conserved vehicle for pathological protein transmission and deliver an immediately translatable pharmacological strategy to arrest disease progression in ALS and FTD.

neuroscience↗

bioRxiv2026

The accumulation of orphan ribosomal proteins is a hallmark of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of poor prognosis, for which age is the strongest risk factor. Despite significant progress in the discovery of ALS-associated mutations, no model explains how such a diversity of mutations converges in a common pathology. In addition, most ALS cases are sporadic and lack known genetic drivers. We recently reported that arginine-rich peptides arising from the C9ORF72 mutation trigger a widespread accumulation of orphan ribosomal proteins (oRP). Here, we show that oRP accumulation is also observed upon expression of other RNA-related ALS mutations, such as hnRNPA2D290V and TDP-43A315T, as well as upon exposure to the ALS-related neurotoxin {beta}-N-methylamino-L-alanine (BMAA). Furthermore, the transcriptional signature of patients with sporadic ALS resembles that of Diamond-Blackfan anemia (DBA), a known ribosomopathy. Supporting the usefulness of our in vitro data, a transcriptional signature defined from these models provides diagnostic and prognostic value in ALS patients. We propose that the accumulation of oRPs due to dysfunctional ribosome biogenesis is a molecular hallmark of ALS that can contribute to the progressive loss of motor neurons in the disease.

neuroscience↗

bioRxiv2026

A chemical inducer of ribophagy limits the toxicity of ALS-related arginine-rich peptides

C9ORF72 intronic repeat expansions are the most frequent mutation found in Amyotrophic Lateral Sclerosis (ALS), producing toxic arginine-rich dipeptides (DPR) that disrupt RNA metabolism and trigger the accumulation of orphan ribosomal proteins (RP). Through a large phenotypic chemical screen, we identified "SALSa", a novel compound that mitigates DPR toxicity. Mechanistically, SALSa acts as a chemical inducer of ribophagy, a specialized form of autophagy that promotes RP clearance. Interestingly, this effect is unrelated to mTOR inhibition, the main regulator of autophagy. In contrast, this is due to an effect of the drug in ribosome biogenesis, which triggers a protective response to clear defective ribosomes. Accordingly, SALSa accumulates in nucleoli and perturbs the final steps of rRNA maturation. SALSa reduces DPR toxicity in differentiated neurons and significantly extends lifespan in a Drosophila melanogaster model of C9ORF72 ALS. These findings suggest that stimulating ribophagy could be beneficial for pathologies associated to dysfunctional ribosome biogenesis, including C9ORF72 ALS.

molecular biology↗

bioRxiv2024

GRK2-mediated AKT activation controls cell cycle progression and G2-checkpoint in a p53-dependent manner

Cell cycle checkpoints, activated by stressful events, halt the cell cycle progression, and prevent the transmission of damaged DNA. These checkpoints prompt cell repair but also trigger cell death if damage persists. Decision-making between these responses is multifactorial and context-dependent, with the tumor suppressor p53 playing a central role. In many tumor cells, p53 alterations lead to G1/S checkpoint loss, rendering cell viability dependent on the strength of the G2 checkpoint through mechanisms not fully characterized. Cells with a strong pro-survival drive can evade cell death despite substantial DNA lesions. Deciphering the integration of survival pathways with p53-dependent and -independent mechanisms governing the G2/M transition is crucial for understanding G2 arrest functionality and predicting tumor cell response to chemotherapy. The serine/threonine kinase GRK2 emerges as a signaling node in cell cycle modulation. In cycling cells, but not in G2 checkpoint-arrested cells, GRK2 protein levels decline during G2/M transition through a process triggered by CDK2-dependent phosphorylation of GRK2 at the S670 residue and Mdm2 ubiquitination. We report now that this downmodulation in G2 prevents the unscheduled activation of the PI3K/AKT pathway, allowing cells to progress into mitosis. Conversely, higher GRK2 levels lead to tyrosine phosphorylation by the kinase c-Abl, promoting the direct association of GRK2 with the p85 regulatory subunit of PI3K and AKT activation in a GRK2 catalytic-independent manner. Hyperactivation of AKT is conditioned by p53s scaffolding function, triggering FOXO3a phosphorylation, impaired cyclin B1 accumulation, and CDK1 activation, causing a G2/M transition delay. Upon G2 checkpoint activation, GRK2 potentiates early arrest independently of p53 through AKT activation. However, its ability to overcome the G2 checkpoint in viable conditions depends on p53. Our results suggest that integrating the GRK2/PI3K/AKT axis with non-canonical functions of p53 might confer a survival advantage to tumor cells.

molecular biology↗

bioRxiv2023

Nucleolar stress caused by arginine-rich peptides triggers a ribosomopathy and accelerates ageing in mice

Nucleolar stress (NS) has been associated to several age-related diseases such as cancer or neurodegeneration. To investigate the mechanisms of toxicity triggered by NS, we here used (PR)n arginine-rich peptides that are found in patients of some neurodegenerative diseases. Although these peptides accumulate at nucleoli and generate NS, how this translates into cellular toxicity is poorly understood. We here reveal that whereas (PR)n expression leads to an overall decrease in protein abundance, this occurs concomitant with an accumulation of free ribosomal (r) proteins in the cytoplasm, a hallmark of ribosomopathies. Conversely, cells with acquired resistance to (PR)n peptides present global downregulation of r-proteins and low levels of mTOR signaling. In mice, systemic expression of (PR)97 drives widespread NS and accelerated ageing, which is associated to an increased expression of r-proteins and mTOR hyperactivation. Furthermore, the reduced lifespan of (PR)97-expressing mice was alleviated by the mTOR inhibitor rapamycin. Importantly, we show that the generalised accumulation of free r-proteins is a common outcome in response to chemical or genetic perturbations that trigger NS, such as Actinomycin D, TIF-IA depletion, or the expression of mutant HMGB1 variants recently associated to rare human diseases. Together, our study presents in vivo evidence supporting the role of NS as a driver of ageing, and provides a general framework to explain the toxicity caused by NS in mammalian cells.

molecular biology↗

bioRxiv2023

The nucleolar aberrancies that drive ribosome impairment induced by RNA binding proteins are hallmarks of aging

The nucleolus is a dynamic structure where ribosome subunits are produced. Indeed, nucleoli respond to any change in cellular homeostasis by altering the rate of ribosome biogenesis, thus working as a stress sensor. Therefore, an imbalance in ribosome biogenesis promotes changes in morphology and function and can evoke a nucleolar stress response. Changes in the structure and composition of nucleoli impair ribosome biogenesis and have been described as nucleolar stress, a mechanism related to aging and cancer. Here, we show the role of the RNA binding protein Hnrnpk in nucleolar dynamics and ribosome function. Hnrnpk is a ribonucleoprotein in charge of escorting nascent transcripts to its processing and nuclear export to ribosomes. When Hnrnpk is overexpressed, the nucleolus is altered and shows stress-like phenotype, with accumulation and delocalization of components such as Ncl, driving ribosome biogenesis impairment and halting protein translation. Nucleolin haploinsufficiency is correlated with enlarged nucleoli, increased ribosome components and translation and induces a reduction in lifespan. Thus, gain of Ncl generated by Hnrnpk overexpression can cause ribosome biogenesis defects associated with ribosome impairment leading to ribosomopathies and bone marrow failure syndrome. Aging and bone marrow failure share common biological hallmarks. Indeed, Hnrnpk overexpression and nucleolar stress trigger cell cycle arrest and senescence of the cells, a feature of both processes. Together, these findings support the idea that nucleolar abnormalities contribute to ribosome impairment, thus triggering the onset of hematopoiesis and the aging process. Here, we decipher a novel master regulator of this mechanism: Hnrnpk.

molecular biology↗

bioRxiv2020

A chemical screen identifies a link between lipid metabolism and mRNA translation

mRNA translation is one of the most energy-demanding processes for living cells, alterations of which have been frequently documented in human disease. Using recently developed technologies that enable image-based quantitation of overall translation levels, we here conducted a chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. Consistent with current knowledge, inhibitors of the mTOR signaling pathway were the most represented class among translation suppresors. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum, which activates the integrated stress response (ISR). Accordingly, the impact of SPHK inhibitors on translation is alleviated by the concomitant inhibition of ISR kinases. On the other hand, and despite the large number of molecules tested, our study failed to identify chemicals capable of substantially increasing mRNA translation, raising doubts on to what extent translation can be supra-physiologically stimulated in mammalian cells. In summary, our study provides the first comprehensive characterization of the effect of known drugs on protein translation and has helped to unravel a new link between lipid metabolism and mRNA translation in human cells.

molecular biology↗

bioRxiv2018

DNA and RNA binding mediate the toxicity of arginine-rich peptides encoded by C9ORF72 GGGGCC repeats

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic-lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids such as RNA transcription, translation, splicing and degradation or DNA replication and repair are impaired by the presence of the CPP. Interestingly, the effects of (PR)n are fully mimicked by PROTAMINE, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated to the onset of ALS.

neuroscience↗

bioRxiv2018

USP7 couples DNA replication termination to mitotic entry.

To ensure a faithful segregation of chromosomes, DNA must be fully replicated before mitotic entry. However, how cells sense the completion of DNA replication and to what extent this is linked to the activation of the mitotic machinery remains poorly understood. We previously showed that USP7 is a replisome-associated deubiquitinase with an essential role in DNA replication. Here, we reveal that USP7 inhibition leads to the ubiquitination of MCM7, a hallmark of DNA replication termination. In addition, USP7 inhibition leads to the ubiquitination of additional replisome components such as POLD1, which are displaced from replisomes. Surprisingly, this premature termination of DNA replication occurs concomitant to a generalized activation of CDK1 throughout the entire cell cycle, which impairs chromosome segregation and is toxic for mammalian cells. Accordingly, the toxicity of USP7 inhibitors is alleviated by CDK1 inhibition. Our work sheds light into the mechanism of action of USP7 inhibitors and provides evidence to the concept that DNA replication termination is coupled to the activation of the mitotic program.

cell biology↗