Biology subjects

Pistello, M.

Publications and source records attributed to Pistello, M..

8 recordsLinked to original sources

bioRxiv2026

Peroxisome dynamics during HSV-1 life cycle in human neurons

HSV-1 is increasingly implicated in Alzheimers disease, yet the mechanisms by which it reshapes neuronal metabolism remain incompletely understood. Here, we demonstrate that HSV-1 co-opts peroxisomal biogenesis and lipid metabolic pathways to promote its replication across human neuronal models. In SH-SY5Y cells, infection triggers a marked expansion of the peroxisomal compartment and alters organelle morphology through upregulation of PGC-1 and PEX13/14/19. Pharmacological stimulation of peroxisome proliferation enhances viral production, whereas inhibition of PEX3-PEX19-dependent biogenesis almost completely suppresses infection. Lipidomic profiling reveals a selective increase in peroxisome-derived plasmalogens and sphingolipids, supporting a role for peroxisomes as a metabolic hub for viral envelopment. This remodeling is recapitulated in hiPSC-derived neurons and human brain organoids, where it is strictly dependent on productive replication and re-emerges upon viral reactivation, but not during latency. Collectively, these findings identify peroxisomes as essential replication-permissive organelles exploited by HSV-1 and suggest that recurrent virus-driven peroxisomal and ether-lipid reprogramming may contribute to neuronal vulnerability in neurodegenerative disease.

microbiology↗

bioRxiv2024

A Transfection-Free Approach of Gene Editing via a gold-based nanoformulation of the Cas9 protein

In recent years, the CRISPR/Cas9 technology has emerged as a highly efficient tool for cell gene editing. However, the delivery of the CRISPR/Cas9 system into cells remains a significant challenge, drastically limiting in vivo gene therapy applications. In this study, we present a transfection/transduction-free tool for intracellular delivery of the Cas9:gRNA ribonucleoprotein. The Cas9 enzyme is conjugated to a 12 nm gold nanoparticle through affinity binding between the 6x His-tag of the protein and the NTA-Ni{superscript 2}LJ groups on the nanoparticles. This link chemistry allows a fine control of the density of the enzymes decorating the particle surface, the orientation of the bonding and the stability of the interaction. Importantly, the surface chemistry of this nanoformulation has been precisely engineered to modulate the cellular internalization and localization. Thanks to this approach of precision chemistry, this nanoformulation demonstrated the ability to spontaneously enter human melanoma cells as monodispersed particles that localize in cell cytoplasm, endosomes, and nucleus. It also shows effective gene editing efficiency similarly to conventional transfection tools. This gold-based formulation of Cas9 represents a ready-to-use biotech editing tool, and a promising solution for direct in vivo gene editing applications.

molecular biology↗

bioRxiv2024

N-acylethanolamine acid amidase inhibition reduces SARS-CoV-2 infection in Human Precision cut-lung slices and downregulates NF-kappaB signalling.

Like other positive-sense RNA viruses, SARS-CoV-2 manipulates host lipid metabolism to facilitate its replication by enhancing lipogenesis and lipid droplet formation. In doing so, SARS-CoV-2 infection perturbs bioactive lipid levels associated with the inflammatory response. One of these, Palmitoylethanolamide (PEA) is suppressed during SARS-CoV-2 infection since it activates the Peroxisome Proliferator-Activated Receptor- (PPAR-), a transcription factor that suppresses the nuclear factor-B (NF-{kappa}B), which is mandatory to sustain SARS-CoV-2 replication. PEA levels are regulated by N-acylethanolamine acid amidase (NAAA), a lysosomal enzyme responsible for catalysing the breakdown of PEA. We hypothesized that NAAA inhibition might interfere with SARS-CoV-2 replication since it will lead PEA to accumulate, activating PPAR- and, consequently, suppressing NF-{kappa}B. Our results reveal that genetic or chemical ablation of NAAA significantly suppresses SARS-CoV-2 replication by three log10 in human-derived precision-cut lung slices. Therefore, we investigated whether inhibiting NAAA could influence NF-{kappa}B activation through the activation of PPAR-. We observed PPAR- increased expression in NAAA-/-cells, while PPAR- expression remained low in infected parental cells. As expected, the elevated PPAR- expression correlated with a parallel reduction in NF-{kappa}B activation when NAAA is ablated. These findings underscore NAAA as an essential host factor for SARS-CoV-2 replication and propose a potential mechanism of action rooted in the attenuation of NF-{kappa}B activation during viral replication. Author summaryOver the past three years, COVID-19 has claimed nearly 7 million lives worldwide, prompting extensive efforts to find effective treatments. While RNA-based vaccines have been developed rapidly, they alone have not completely halted the spread of the virus, making the search for antiviral therapies crucial. One promising approach targets the anti-inflammatory lipid PEA, which has shown some success in COVID-19 clinical trials. PEA is quickly degraded by the enzyme NAAA. Researchers have found that inhibiting NAAA can enhance and prolong PEA anti-inflammatory effects. NAAA inhibitors have already shown effectiveness in reducing chronic pain and lung inflammation in animal models and have also been effective against Zika virus replication. Our research focused on testing the NAAA inhibitor ARN726 against SARS-CoV-2. In human lung cells and lung tissue samples, ARN726 significantly reduced SARS-CoV-2 replication and inflammation. We discovered that this inhibition suppresses the NF-{kappa}B pathway, which the virus uses to fuel its replication and sustain Cytokine storm. Overall, our findings suggest that NAAA inhibitors like ARN726 could be repurposed to combat COVID-19 and potentially other coronaviruses, offering a novel and effective antiviral strategy.

microbiology↗

bioRxiv2023

SARS-CoV-2 infection leads to Tau pathological signature in neurons

BackgroundThe coronavirus disease 19 (COVID-19) has represented an issue for global health since its outbreak in March 2020. It is now evident that the SARS-CoV-2 infection results in a wide range of long-term neurological symptoms and is worryingly associated with the aggravation of Alzheimers disease. Little is known about the molecular basis of these manifestations. MethodsSeveral SARS-CoV-2 strain variants were used to infect SH-SY5Y neuroblastoma cells and K18-hACE C57BL/6J mice. The Tau phosphorylation profile and aggregation propensity upon infection were investigated using immunoblot and immunofluorescence on cellular extracts, subcellular fractions, and brain tissue. The viral proteins Spike, Nucleocapsid, and Membrane were overexpressed in SH-SY5Y cells and the direct effect on Tau phosphorylation was checked using immunoblot experiments. ResultsUpon infection, Tau is phosphorylated at several pathological epitopes associated with Alzheimers disease and other tauopathies. Moreover, this event increases Taus propensity to form insoluble aggregates and alters its subcellular localization. ConclusionsOur data support the evidence that SARS-CoV-2 infection in the Central Nervous System triggers downstream effects altering Tau function, eventually leading to the impairment of neuronal function.

neuroscience↗

bioRxiv2023

The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.

microbiology↗

bioRxiv2022

N-acylethanolamine acid amide hydrolase is a novel target for drugs against SARS-CoV-2 and Zika virus

Several compounds have been tested against SARS-CoV-2; at present, COVID-19 treatments decrease the deleterious inflammatory response and acute lung injury. However, the best therapeutic response would be expected by combining anti-inflammatory properties, while concomitantly blocking viral replication. These combined effects should drastically reduce both infection rate and severe complications induced by novel SARS-CoV-2 variants. Therefore, we explored the antiviral potency of a class of anti-inflammatory compounds that inhibit the N-Acylethanolamine acid amidase (NAAA). This enzyme catalyzes the hydrolysis of palmitoylethanolamide (PEA), a bioactive lipid that mediates anti-inflammatory and analgesic activity through the activation of peroxisome proliferator receptor- (PPAR-). Similarly, this pathway is likely to be a significant target to impede viral replication since PPAR- activation leads to dismantling of lipid droplets, where viral replication of Flaviviruses and Coronaviruses occurs. Here, we show that either genetic or pharmacological inhibition of the NAAA enzyme leads to five-fold reduction in the replication of both SARS-CoV-2 and ZIKV in various cell lines. Once NAAA enzyme is blocked, both ZIKV and SARS CoV-2 replication decrease, which parallels a sudden five-fold decrease in virion release. These effects induced by NAAA inhibition occurs concomitantly with stimulation of autophagy during infection. Remarkably, parallel antiviral and anti-inflammatory effects of NAAA antagonism were confirmed in ex-vivo experiments, within SARS-CoV-2 infected human PBMC cells, in which both viral genomes and TNF- production drop by ~60%. It is known that macrophages contribute to viral spread, excessive inflammation and macrophage activation syndrome that NAAA inhibitors might prevent, reducing the macrophage-induced acute respiratory distress syndrome and subsequent death of COVID-19 patients.

microbiology↗

bioRxiv2021

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells

We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results suggest that in these cells the variant of concern B.1.1.7, (aka Alpha variant), became the predominant circulating variant in several countries by a clear transmission advantage. In fact, in these cells B.1.1.7 outcompetes its ancestor B.1.177 in terms of a much faster kinetics of entry. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the main role of clathrin as mediator of late-entry endocytosis, reconciling it with the membrane localization of the ACE2 receptor previously attributed to caveolin-enriched rafts. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

biophysics↗

bioRxiv2020

HIV-1 provirus excised by single CRISPR/Cas9 RNA guide persists in host cell and may be reactivated

Gene editing may be used to cut out the human immunodeficiency virus type-1 (HIV-1) provirus from the host cell genome and eradicate infection. Here, using cells acutely or latently infected by HIV and treated with long terminal repeat-targeting CRISPR/Cas9, we show that the excised HIV provirus persists for a few weeks and, by means of HIV Integrase, rearranges in circular molecules. Circularization and integration restore proviral transcriptional activity that is enhanced in the presence of exogenous Tat and Rev or tumor necrosis factor-, respectively, in acutely or latently infected cells. Although confirming that gene editing is a powerful tool to eradicate HIV infection, this work highlights that, to achieve this goal, the provirus has to be cleaved in several pieces and the infected cells treated with antiviral therapy before and after editing.

microbiology↗