Biology subjects

Bruno, L.

Publications and source records attributed to Bruno, L..

9 recordsLinked to original sources

bioRxiv2026

Divergent consequences of PSEN1 knockout and PSEN2 knockout in stem cell derived models of the brain

{gamma}-secretase is a multi-subunit enzyme complex responsible for cleaving hundreds of substrates in diverse cellular contexts. Variation in subunit composition - including the use of alternate catalytic subunits Presenilin 1 (PSEN1) and Presenilin 2 (PSEN2) - results in diverse {gamma}-secretase complexes. Point mutations in PSEN1 and PSEN2 cause familial forms of Alzheimers disease, while loss-of-function mutations in the {gamma}-secretase subunits PSEN1, PSENEN and NCSTN cause acne inversa. To advance therapeutic strategies targeting {gamma}-secretase in Alzheimers disease, a better understanding of individual {gamma}-secretase complexes is required. In this study, we used CRISPR-Cas9 genome engineering to generate PSEN2-knockout iPSCs in order to compare the consequence of PSEN2 knockout versus PSEN1 knockout in iPSC-derived brain cells. In contrast to PSEN1-knockout, PSEN2-knockout did not alter APP cleavage or A{beta} generation in iPSC-neurons, nor did it disrupt Nicastrin maturation. Similarly, PSEN2-knockout had little impact on TREM2 processing in iPSC-microglia. Instead, our data indicate that loss of PSEN2 primarily impacts the endo-lysosomal system in iPSC-neurons, causing an accumulation of early endosome markers and a reduction in lysosomal markers - phenotypes not observed in PSEN1-knockout neurons. Taken together, these findings highlight distinct and non-redundant functions of PSEN1 and PSEN2 in human brain cells, reinforcing findings in animal models and subcellular localisation studies. This work advances our understanding of distinct {gamma}-secretase complex functions and provides insights that will support future therapeutic efforts to inhibit, modulate or stabilise {gamma}-secretase.

neuroscience↗

bioRxiv2024

DNA topoisomerase I acts as supercoiling sensor for transcription elongation in E. coli

When DNA is transcribed to RNA, the DNA double helix is constantly unwound and rewound to provide access for RNA polymerase (RNAP). This induces DNA supercoiling as a function of transcript length due to over- and under-twisting of the DNA downstream and upstream of RNAP, respectively. Using single-particle cryo-EM and in vivo assays we investigated the relationship between bacterial RNAP and DNA Topoisomerase I (TopoI), which removes negative supercoils accumulating upstream of RNAP. TopoI binds to relaxed DNA upstream of RNAP in a manner suggesting a sensory role awaiting the formation of negative supercoils and involving a conformational switch in the functional domains of TopoI. On DNA substrates mimicking negatively supercoiled DNA, TopoI threads one strand into the active site for cleavage while binding the complementary strand with an auxiliary domain. We propose a comprehensive model for DNA relaxation in the context of a transcribing RNAP.

molecular biology↗

bioRxiv2024

Non-CG DNA methylation modulates hypocotyl elongation during thermormorphogenesis

Plants adapt to warm environments through physiological and morphological changes termed thermomorphogenesis, which involve transcriptional reprogramming exerted mainly by PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Fluctuating temperatures can also influence the patterns of cytosine DNA methylation, thereby influencing gene expression. However, whether these epigenetic changes provide an adaptative advantage remains unclear. Here, we provide evidence that DNA methylation is required to regulate thermomorphogenesis. Hypomethylated drm1 drm2 cmt3 mutants or seedlings treated with 5-azacytidine to block DNA methylation exhibit reduced hypocotyl growth at warm temperatures, primarily due to impaired cell elongation. Moreover, DNA hypomethylation compromises auxin biosynthesis and transport in response to warmth, partially by reducing PIF4 protein levels. Notably, the loss of DNA methylation leads to increased expression of SUPPRESSOR OF drm1 drm2 cmt3 (SDC), which in turn restricts hypocotyl elongation during thermomorphogenesis. Finally, we demonstrate that DNAme regulates the inhibition of SDC expression to promote gibberellin biosynthesis. Our findings underscore the critical role of DNA methylation in modulating gene expression in response to temperature fluctuations and provide new insights into the epigenetic regulation of thermomorphogenesis. HighlightsDNA methylation regulates the expression of key genes involved in auxin and gibberellin metabolism, to ensure hypocotyl growth in response to warm temperatures.

plant biology↗

bioRxiv2024

Ongoing local adaptation to climate change in yarrow (Achillea millefolium L.)

Climate change threatens biodiversity as populations can persist if they migrate or adapt to the rapidly changing conditions of the Anthropocene. However, little is known about the persistence of plant populations under the long-term trends of increasing temperature and drought. We explore whether historical populations of yarrow (Achillea millefolium L.) surveyed in the 1920s have persisted locally or undergone local extinction after 100 years. We resurveyed historical sites spanning a broad climatic gradient (from 1 m to 3,200 m a.s.l.), examined to what extent plant growth signature (height) has changed over time, and analyzed metabolic diversity. Nine out of ten populations persisted locally and showed phenotypic and metabolic differentiation. The only population potentially extirpated is that of the hottest and driest site. Populations from warm sites in coastal and mountain regions had grown taller than 100 years ago. In contrast, populations from dry sites in lowlands and foothills became shorter. Furthermore, we document differentiation in metabolic diversity involving plant defenses and signaling. Ongoing local adaptation is constrained by changing both temperature and precipitation as well as by biotic interactions. Preserving locally adapted populations and metabolic diversity is key for conservation efforts.

evolutionary biology↗

bioRxiv2024

Ecotin protects Salmonella Typhimurium against the microbicidal activity of host proteases

Salmonella enterica serovar Typhimurium causes acute diarrhea upon oral infection in humans. The harsh and proteolytic environment found in the gastrointestinal tract is the first obstacle that these bacteria face after infection. However, the mechanisms that allow Salmonella to survive the hostile conditions of the gut are poorly understood. The ecotin gene is found in an extensive range of known phyla of bacteria and it encodes a protein that has been shown to inhibit serine proteases. Thus, in the present work we studied the role of ecotin of Salmonella Typhimurium in host-pathogen interactions. We found that Salmonella Typhimurium {Delta}ecotin strain exhibited lower inflammation in a murine model of Salmonella induced colitis. The {Delta}ecotin mutant was more susceptible to the action of pancreatin and purified pancreatic elastase. In addition, the lack of ecotin led to impaired adhesion to Caco-2 and HT-29 cell lines, related to the proteolytic activity of brush border enzymes. Besides, {Delta}ecotin showed higher susceptibility to lysosomal proteolytic content and intracellular replication defects in macrophages. In addition, we found Ecotin to have a crucial role in Salmonella against the microbicide action of granules released and neutrophil extracellular traps from human polymorphonuclear leukocytes. Thus, the work presented here highlights the importance of ecotin in Salmonella as countermeasures against the host proteolytic defense system. IMPORTANCEThe gastrointestinal tract is a very complex and harsh environment. Salmonella is a successful food borne pathogen, but little is known about its capacity to survive against the proteolysis of the gut lumen and intracellular proteases. Here, we show that Ecotin, a serine protease inhibitor, plays an important role in protecting Salmonella against proteases present at different sites encountered during oral infection. Our results indicate that Ecotin is an important virulence factor in Salmonella, adding another tool to the wide range of features this pathogen uses during oral infection.

microbiology↗

bioRxiv2023

Fully Automated Sequential Immunofluorescence (seqIF) for Hyperplex Spatial Proteomics

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 hours, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling quantitative downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

cancer biology↗

bioRxiv2020

Following the footprints of variability during filopodia growth

Filopodia are actin-built finger-like dynamic structures that protrude from the cell cortex. These structures can sense the environment and play key roles in migration and cell-cell interactions. The growth-retraction cycle of filopodia is a complex process exquisitely regulated by intra- and extra-cellular cues, whose nature remains elusive. Filopodia present wide variation in length, lifetime and growth rate. Here, we investigate the features of filopodia patterns in fixed prostate cancer cells by confocal microscopy. Analysis of almost a thousand filopodia suggests the presence of two different populations: one characterized by a narrow distribution of lengths and the other with a much more variable pattern with very long filopodia. We explore a stochastic model of filopodia growth which takes into account diffusion and reactions involving actin and the regulatory proteins formin and capping, and retrograde flow. Interestingly, we found an inverse dependence between the filopodial length and the retrograde velocity. This result led us to propose that variations in the retrograde velocity could explain the experimental lengths observed for these tumor cells. In this sense, one population involves a wider range of retrograde velocities than the other population, and also includes low values of this velocity. It has been hypothesized that cells would be able to regulate retrograde flow as a mechanism to control filopodia length. Thus, we propound that the experimental filopodia pattern is the result of differential retrograde velocities originated from heterogeneous signaling due to cell-substrate interactions or prior cell-cell contacts.

biophysics↗

bioRxiv2020

Epigenetic change induced by in utero dietary challenge provokes phenotypic variability across multiple generations of mice

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, histone H3K9me3, H3K27me3, H4K16ac and DNA methylation1-3. In higher vertebrates, epidemiological and experimental evidence supports similar trans-generational effects4,5 although the mechanisms that underpin these are incompletely understood6-9. We generated a luciferase reporter knock-in mouse for the imprinted Dlk1 locus, to visualise and track epigenetic fidelity across generations. We showed that exposure to high-fat diet (HFD) in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 allele in offspring, coincident with increased DNA methylation at the Dlk1 sDMR. Interestingly, maternal Dlk1 mis-expression was also evident in the next generation (F2), exclusively in animals derived from F1-exposed females. Oocytes from these females showed altered microRNA and gene expression, without any major changes in underlying DNA methylation, and correctly imprinted Dlk1 expression resumed in subsequent generations (F3 onwards). Our results reveal how canonical and non-canonical imprinting mechanisms enable the foetal epigenome to adapt to in utero challenge to modulate the properties of two successive generations of offspring.

developmental biology↗

bioRxiv2020

Early defects in lysosomal storage diseases disrupt excitatory synaptic transmission

At least two thirds of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPS IIIC, Sanfilippo disease type C) caused by mutations in the HGSNAT gene and lysosomal storage of heparan sulfate the major burden is progressive and severe neuropsychiatric problems, mental retardation, and dementia though to be mainly mediated by neurodegeneration. HGSNAT knockout mice match human clinical phenotype and develop hyperactivity followed by memory impairment and death. In order to understand whether early clinical symptoms in MPS IIIC mice occurring before the onset of massive neurodegeneration are caused by neuronal dysfunction we studied synaptic transmission and morphology in cultured hippocampal and CA1 pyramidal neurons of MPSIIIC mice. Synaptic spines were also studied in other mouse LSD models and postsynaptic densities in post-mortem cortices of human neurological MPS patients. Cultured hippocampal and CA1 pyramidal neurons of MPS IIIC mice showed a drastic decrease or abnormal distribution of multiple pre- and postsynaptic proteins that could be rescued in vitro and in vivo by virus-mediated gene correction. Dendritic spine densities were immature in cultured hippocampal MPS IIIC mouse neurons and reduced in pyramidal neurons of mouse models of MPS IIIC and other (Tay-Sachs, sialidosis) LSD starting from postnatal day 10. MPS IIIC neurons presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganised microtubule network and partially impaired axonal transport of synaptic proteins. Postsynaptic densities were also reduced in post-mortem cotrees of human MPS I, II, IIIA, C and D patients suggesting that the pathology is common for these neurological LSD. Together, our results demonstrate that lysosomal storage causes alterations in synaptic structure and abnormalities in neurotransmission originating from disrupted vesicular transport and preceding the first cognitive symptoms and suggest drugs known to affect synaptic transmission can be potentially applied to treat behavioral and cognitive defects in neurological LSD patients.

neuroscience↗