Biology subjects

Lombardi, V.

Publications and source records attributed to Lombardi, V..

8 recordsLinked to original sources

bioRxiv2026

Sustained Activation of a Defensive Behavior-Related Circuit Elicits a distinct Homeostatic Sleep Response

Animals and humans often forgo sleep in response to other physiological needs. Prolonged wakefulness leads to a compensatory homeostatic sleep response involving enhanced amounts of sleep and electroencephalographic delta (0.5-4 Hz) power, yet how the type of preceding wakefulness and brain activity affects this response is poorly understood. Here we compared the pattern of brain activity and the homeostatic sleep response following sustained wakefulness induced by either gentle handling (GH), a widely used form of sleep deprivation in rodents; or optogenetic stimulation of neurons involved in avoidance/defensive responses, basal forebrain (BF) vesicular glutamatergic transporter type 2 (vGluT2) neurons. Local infusion of ionotropic glutamate receptor antagonists partially attenuated the wake-inducing effect of vGluT2-stimulation, but not that of GH, suggesting mediation by other non-glutamatergic BF subtypes. These antagonists also tended to produce more NREM sleep and a shorter NREM latency in the recovery period following vGluT2-stimulation. Both manipulations increased high theta (7-9 Hz) during wakefulness, an effect partially blocked by local infusion of the glutamate antagonist cocktail. Enhanced NREM sleep time and NREM delta power in the recovery period were more pronounced following GH than following vGluT2 stimulation. Whole-brain cFos immunohistochemistry revealed stronger neuronal activity in brain regions involved in stress and avoidance/defensive behaviors with vGluT2 stimulation, compared to GH. Our results demonstrate that activation of a brain system involved in defensive behavior (BF vGluT2) elicits a distinct and diminished homeostatic sleep response compared to GH, potentially an evolutionary sleep adaptation which would maintain vigilance in the face of an ongoing threat.

neuroscience↗

bioRxiv2025

PatientProfiler: A network-based approach to personalized medicine

Deciphering the intricate mechanisms underlying reprogramming in cancer cells is a crucial challenge in oncology as it holds the key to advance our ability to diagnose and treat cancer patients. For this reason, comprehensive and patient-specific multi-omic characterization of tumor specimens has become increasingly common in clinical practice. While these efforts have advanced our understanding of the molecular mechanisms underlying breast cancer progression, the identification of personalized therapeutic approaches remains a distant goal. The main shortcoming is the absence of a robust computational framework to integrate and interpret the available multi-dimensional data and to drive translational solutions. To fill this gap, we developed PatientProfiler, a computational pipeline that leverages causal interaction data, annotated in our in-house manually-curated resource, SIGNOR, to address how the genetic and molecular background of single patients contributes to the establishment of a malignant phenotype. PatientProfiler is an open-source, R-based package composed of several functions that allows multi-omic data analysis and standardization, generation of patient-specific mechanistic models of signal transduction, and extraction of network-based prognostic biomarkers. To benchmark the tool, we retrieved genomic, transcriptomic, (phospho)proteomic, and clinical data derived from 122 treatment-naive breast cancer biopsies, available at the CPTAC portal. Thanks to this approach, we identified patient-specific mechanistic models (one patient, one network) that recapitulate dysregulated signaling pathways in breast cancer. This collection of models provides valuable insights into the underlying mechanisms of tumorigenesis and disease progression. Moreover, in-depth topological exploration of these networks has allowed us to define seven communities (subnetworks), each associated with a unique transcriptomic signature and a distinct prognostic value. In summary, our work demonstrates that PatientProfiler is a tool for patient-specific network analysis, advancing personalized medicine towards the identification of actionable biomarkers and tailored therapeutic strategies.

systems biology↗

bioRxiv2024

An integrated picture of the structural pathways controlling the heart performance

Regulation of heart function is attributed to a dual filament mechanism: (i) the Ca2+-dependent structural changes in the regulatory proteins of the thin, actin-containing filament making actin available for myosin motor attachment1, and (ii) the release of motors from their folded (OFF) state on the surface of the thick filament2 allowing them to attach and pull the actin filament. Thick filament mechanosensing is thought to control the number of motors switching ON in relation to the systolic performance3, but its molecular basis is still unknown. Here high spatial resolution X-ray diffraction data from electrically paced rat trabeculae and papillary muscles call for a revision of the mechanosensing hypothesis and provide a molecular explanation of the modulation of heart performance also in light of the recent cryo-EM thick filament structure4, 5. We find that upon stimulation titin activation6 triggers structural changes in the thick filament that switch motors ON throughout the filament within [~][1/2] the maximum systolic force. These structural changes also drive MyBP-C N-terminus to bind actin7 promoting first motor attachments from the central 1/3 of the half-thick filament. Progression of attachments towards the periphery of half-thick filament with increase in systolic force is carried on by near-neighbour cooperative thin filament activation by attached motors8. The identification of the roles of MyBP-C, titin, thin and thick filaments in heart regulation enables their targeting for potential therapeutic interventions.

physiology↗

bioRxiv2021

Titin switches from an extensible spring to a mechanical rectifier upon muscle activation

In contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm-1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.

physiology↗

bioRxiv2020

A myosin II-based nanomachine devised for the study of Ca2+-dependent mechanisms of muscle regulation

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine, made by an ensemble of eight HMM myosin fragments from rabbit psoas muscle carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca2+-regulated actin binding protein) to a bead trapped by a Dual Laser Optical Tweezers. The application of the original version of the nanomachine to investigation of the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them on at a time, was anyway prevented by the impossibility to preserve Ca2+ as a free parameter. Here the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined either in the absence or in the presence of 0.1 mM Ca2+ (the concentration required for BTA preparation with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.

biophysics↗

bioRxiv2020

Analysis of circulating protein aggregates reveals pathological hallmarks of amyotrophic lateral sclerosis

Blood-based biomarkers can be informative of brain disorders where protein aggregation play a major role. The proteome of plasma and circulating protein aggregates (CPA) reflect the inflammatory and metabolic state of the organism and can be predictive of system-level and/or organ-specific pathologies. CPA are enriched with heavy chain neurofilaments (NfH), key axonal constituents involved in brain aggregates formation and biomarkers of the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Here we show that CPA and brain protein aggregates (BPA) from ALS differ in protein composition and appear as a combination of electron-dense large globular and small filamentous formations on transmission electron microscopy. CPA are highly enriched with proteins involved in the proteasome and energy metabolism. Compared to the human proteome, proteins within aggregates show distinct and tissue-dependent chemical features of aggregation propensity. The use of a TMTcalibrator proteomics workflow with ALS brain as calibrant reveals 4973 brain-derived low-abundance proteins in CPA, including the products of translation of 24 ALS risk genes. 285 of these (5.7%) are regulated in ALS CPA including FUS (p<0.05). CPA from both ALS and healthy controls affect cell viability when testing endothelial and PC12 neuronal cell lines, while CPA from ALS exert a more toxic effect at lower concentrations. The analysis of resistance to protease enzymes hydrolysis indicates an ALS-specific digestion pattern for NfH using enterokinase. This study reveals how peripheral protein aggregates are significantly enriched with brain proteins which are highly representative of ALS pathology and a potential alternative source of biomarkers and therapeutic targets for this incurable disorder. Significance StatementMolecular mechanism of neurodegeneration like protein aggregation are important brain-specific alterations which need to be addressed therapeutically. Recently described fluid biomarkers of neurodegenerative disorders provide means for stratification and monitoring of disease progression. Here we show that circulating protein aggregates are easily accessible in blood and reproduce important features of brain pathology for an incurable disorder like amyotrophic lateral sclerosis. They represent a source of biomarkers and of novel therapeutics for ALS.

neuroscience↗

bioRxiv2020

Immune reactivity to neurofilaments and dipeptide repeats in ALS progression

ObjectiveTo test antibody response and formation of immune-complexes to neurofilaments and dipeptide-repeats, the translational products of the mutated C9orf72 gene, as potential biomarkers for clinical stratification of amyotrophic lateral sclerosis (ALS). MethodsUsing neurofilament protein isoforms plasma expression as reference, antibodies and immune-complexes against neurofilament-light, medium and heavy chain and poly-(GP)-GR dipeptide-repeats were tested in blood from 105 fast and slow progressing ALS patients, 26 C9orf72 mutation carriers (C9+ve) ALS patients and 77 healthy controls (HC) using single-molecule and immune-capture assays. Longitudinal antibody/immune-complex responses were measured in serial blood samples from 37 (including 11 C9+ve) patients. ResultsAntibodies and immune-complex reactivity was higher in ALS patients than HC, particularly in C9+ve ALS patients, and modestly correlated with total neurofilament protein isoforms (r:0.24 p=0.002; r:0.18 p=0.02 respectively). Neurofilament-light immune-complexes and neurofilament-heavy antibodies had the best diagnostic performances distinguishing ALS subtypes from HC (AUC=0.68 p<0.01; AUC=0.68 p<0.001 respectively). Neurofilament-light immune-complexes (AUC=0.69 p<0.01) and poly-(GP) dipeptide-repeats antibodies (AUC=0.71 p<0.001) separated C9+ve from C9-ve patients. Multivariate mortality hazard ratio and Kaplan-Meier analyses showed low neurofilament-heavy antibody levels associated with increased survival. Longitudinal analysis identified raising levels of antibodies against neurofilaments in fast progressing ALS and of neurofilament-light immune-complexes in C9+ve patients. InterpretationC9+ve and fast progressing ALS patients have a distinct neurofilament and dipeptide-repeat immuno-phenotype, with increasing levels of blood neurofilament-light immune-complexes and neurofilament antibodies with disease progression. The study of the expression of these biomarkers in the natural history of ALS may shed light on disease initiation and progression and provide novel pharmacodynamic biomarkers in emerging C9orf72 gene silencing therapies.

neuroscience↗

bioRxiv2019

Plasma pNfH differentiate SBMA from ALS

Background and aimSpinal bulbar muscular atrophy (SBMA) is a progressive adult-onset X-linked neuromuscular disease. Although traditionally considered a motor neuron disorder, recent advances have highlighted a primary myopathic component. We evaluated levels of phosphorylated neurofilament heavy chain (pNfH), a known biomarker for neurodegeneration, in SBMA. Materials and methodsWe collected plasma and serum from 46 SBMA, 50 ALS and 50 healthy control cases, alongside with plasma from a mouse model of SBMA (AR100) and littermate controls. We measured pNfH plasma levels using Single molecule array (Simoa), we assessed functional scales and we gathered demographic data. We analysed data using Mann-Whitney U test, Kruskal-Wallis test and Cox regression analysis. ResultsPlasma pNfH levels were significantly increased in ALS, but, intriguingly, there was no change in SBMA. These results were also confirmed in SBMA mice. The ROC curve highlighted that pNfH levels can effectively distinguish between ALS and SBMA (AUC 0.95). ConclusionsUnexpectedly, levels of pNfH are normal in SBMA, whilst they are increased in ALS, and suggest pNfH could serve as a biomarker to differentiate the two diseases. Further, this finding is in agreement with recent evidence showing that primary muscle damage is a crucial feature in SBMA.

neuroscience↗