Biology subjects

Novello, S.

Publications and source records attributed to Novello, S..

6 recordsLinked to original sources

bioRxiv2025

Nuclear ASC speck formation in microglia is associated with inflammasome priming and is exacerbated in LRRK2-G2019S Parkinson disease

Neuroinflammation is increasingly recognized as a central pathological mechanism in Parkinsons disease (PD), a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and variety of motor and non-motor symptoms. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome and its adaptor protein ASC play a critical role in initiating and maintaining inflammatory responses in the central nervous system. Although its acute activation is beneficial for host defense and homeostasis, chronic activation of the inflammasome has been associated with the pathogenesis of PD. Another key contributor to neuroinflammation is the leucine-rich repeat kinase 2 (LRRK2), particularly the G2019S mutation associated with PD, which has been shown to exacerbate inflammatory signaling in microglia and peripheral immune cells. However, the interaction between LRRK2 and the NLRP3 inflammasome pathway remains poorly understood. In this study, we investigated the role of LRRK2-G2019S in the priming and activation dynamics of the NLRP3 inflammasome using mouse primary microglia and human monocyte-derived microglia-like cells (hMDMi). We observed that LRRK2-G2019S microglia exhibit increased expression of NLRP3 under basal conditions and spontaneous formation of ASC specks within the nucleus, an unexpected subcellular location not previously reported in microglia. Interestingly, nuclear ASC specks also formed in wild-type microglia and hMDMi after lipopolysaccharide (LPS) priming but only progressed to cytosolic ASC specks and interleukin-1{beta} release after subsequent exposure to canonical NLRP3 activators. These findings suggest that nuclear ASC specks may represent a primed state of inflammasome activation and propose a novel cellular phenotype associated with LRRK2-G2019S. Altogether, our results reveal a new layer of inflammasome regulation in microglia and implicate LRRK2-G2019S in the promotion of a pro-inflammatory state, which may predispose to chronic neuroinflammation in PD. These findings advance our understanding of glial immune regulation and highlight potential therapeutic targets in PD.

physiology↗

bioRxiv2024

Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

Alpha-synuclein (aSyn) post-translational modifications (PTMs), particularly phosphorylation at serine 129 and C-terminal truncations, are highly enriched in Lewy bodies (LBs), Lewy neurites, and other types of aSyn pathological aggregates in the brain of patients with Parkinsons disease (PD) and other synucleinopathies. However, our knowledge about the precise role of PTMs in regulating the different stages of pathology formation, neurodegeneration, and aSyn pathology spreading remains incomplete. In this work, we applied a systematic approach to address this knowledge gap with an emphasis on mapping and elucidating the role of post-fibrillization C-terminal aSyn truncations in regulating the uptake, processing, seeding activity, and formation of LB-like inclusions and maturations in a well-established neuronal seeding model that recapitulates all the stages leading to LB formation and neurodegeneration. Our work shows that C-terminal cleavage of aSyn fibrils at multiple sites is a conserved process that occurs rapidly after and during the formation of intracellular LB-like aSyn inclusions in all neuronal seeding models. Interestingly, blocking the cleavage of internalized fibrils does not influence their seeding activity, whereas inhibiting the enzymes that regulate the cleavage of newly formed fibrils (e.g., calpains 1 and 2) significantly alters the formation of LB-like inclusions. We also show that C-terminal truncations, in combination with other PTMs, play a crucial role in regulating the interactome and remodeling of newly formed aSyn fibrils, including their shortening, lateral association, and packing during LB formation and maturation. Altogether, our results demonstrate that post-fibrillization C-terminal truncations have a differential role at different stages of aSyn aggregation and pathology formation. These insights, combined with the abundance of truncated aSyn species in LBs, have significant implications in understanding aSyn pathological diversity and developing therapeutic strategies targeting the C-terminus of aSyn or its proteolytic processing.

neuroscience↗

bioRxiv2022

On demand nanoliter sampling probe for collection of brain fluid

Continuous fluidic sampling systems allow collection of brain biomarkers in vivo. Here, we propose a new sampling paradigm, Droplet on Demand (DoD), implemented in a microfabricated neural probe. It allows sampling droplets loaded with molecules from the brain extracellular fluid punctually, without the long transient equilibration periods typical of continuous methods. It uses an accurate fluidic sequence and correct operation is verified by the embedded electrodes. As a proof of concept, we demonstrated the application of this novel approach in vitro and in vivo, to collect glucose in the brain of mice, with a temporal resolution of 1-2 minutes and without transient regime. Absolute quantification of the glucose level in the samples was performed by direct infusion nanoelectrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS). By adjusting the diffusion time and the perfusion volume of DoD, the fraction of molecules recovered in the samples can be tuned to mirror the tissue concentration at accurate points in time. This makes quantification of biomarkers in the brain possible within acute experiments of only 20 to 120 minutes. DoD provides a complementary tool to continuous microdialysis and push-pull sampling probes. The advances allowed by DoD will benefit quantitative molecular studies in the brain, namely for molecules involved in volume transmission or for protein aggregates that form in neurodegenerative diseases over long periods. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/487549v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1edeb56org.highwire.dtl.DTLVardef@f573aforg.highwire.dtl.DTLVardef@7dfd38org.highwire.dtl.DTLVardef@17b7b46_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies

Alpha-synuclein (aSyn) within Lewy bodies, Lewy neurites, and other pathological hallmarks of Parkinsons disease and synucleinopathies have consistently been shown to accumulate in aggregated and phosphorylated forms of the protein, predominantly at Serine 129 (S129). Antibodies against phosphorylated S129 (pS129) have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues. However, most of the antibodies and immunoassays aimed at detecting pS129-aSyn were developed based on the assumption that neighbouring post-translational modifications (PTMs) either do not co-occur with pS129 or do not influence its detection. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal PTMs (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighbouring PTMs. However, consistent with previous reports, most pS129 antibodies showed cross-reactivity towards other proteins and often detected low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or High Molecular Weight aggregates of aSyn. Our observations suggest that the pS129 antibodies do not capture the biochemical and morphological diversity of aSyn pathology. They also underscore the need for more specific pS129 antibodies, more thorough characterization and validation of existing antibodies, and the use of the appropriate protein standards and controls in future studies.

neuroscience↗

bioRxiv2020

The P2X7 receptor localizes to the mitochondria, modulates mitochondrial energy metabolism and enhances physical performance

Basal expression of the P2X7 receptor (P2X7R) improves mitochondrial metabolism, ATP synthesis and overall fitness of immune and non-immune cells. We investigated P2X7R contribution to energy metabolism and subcellular localization in fibroblasts (mouse embryo fibroblasts and HEK293 human fibroblasts), mouse microglia (primary brain microglia and the N13 microglia cell line), and heart tissue. The P2X7R localizes to mitochondria, and its lack a) decreases basal respiratory rate, ATP-coupled respiration, maximal uncoupled respiration, resting mitochondrial potential, mitochondrial matrix Ca2+ level, b) modifies expression pattern of oxidative phosphorylation (OxPhos) enzymes, and c) severely affects cardiac performance. Hearts from P2rx7-deleted versus WT mice are larger, heart mitochondria smaller, and stroke volume (SV), ejection fraction (EF), fractional shortening (FS) and cardiac output (CO), are significantly decreased. Accordingly, physical fitness of P2X7R-null mice is severely reduced. Thus, the P2X7R is a key modulator of mitochondrial energy metabolism and a determinant of physical fitness.

cell biology↗

bioRxiv2019

Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in non-human primates

AO_SCPLOWBSTRACTC_SCPLOWDopaminergic neuronal cell death, associated with intracellular -synuclein (-syn)-rich protein aggregates (termed Lewy bodies), is a well-established characteristic of Parkinsons disease. Much evidence, accumulated from multiple experimental models has suggested that -syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct -syn pathogenic structures derived from PD patients. Unexpectedly, our results show that, in non-human primates, a small amount of singular -syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD as multiple causes can induce similar outcome regarding dopaminergic neurodegeneration.

neuroscience↗