Biology subjects

De Carli, A.

Publications and source records attributed to De Carli, A..

6 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↗

bioRxiv2026

Unconventional Small Molecule MNP-021 Protects Neuronal and Glial Function from Diabetes-Associated Glucotoxicity and Neuroinflammation

BackgroundDiabetes-associated neurodegeneration is amplified by methylglyoxal (MGO)-driven dicarbonyl stress linking hyperglycemia to neuronal insulin resistance and maladaptive neuroinflammation. We tested the neuroprotective activity of MNP-021, a non-electrophilic TRPA1 modulator, in neurons and glial cells in vitro. MethodsSH-SY5Y neurons were pretreated with MNP-021 and challenged with MGO, then profiled by high-content imaging, RNA-seq, Seahorse OCR/ECAR, glycolytic stress assays and AKT/ERK/CREB immunoblotting {+/-} insulin. In parallel, HMC3 glial cells were treated with MNP-021, exposed to LPS/TNF- or A{beta}(25-35) and tested for viability and inflammatory markers by ELISA and qRT-PCR. ResultsMGO increased nucleus-to-cytoplasm area ratio by 49% and dysregulated glucose handling, increasing 2-NBDG uptake by [~]25%, with GLUT1/GLUT4 membrane redistribution; MNP-021 normalized morphology, uptake, and transporter localization without cytotoxicity up to 10 {micro}M. RNA-seq identified 754 MGO-deregulated genes, including ISR/metabolic nodes (GCK, SESN2, PHGDH/PSAT1, PCK2); MNP-021 buffered stress-induced transcription with limited baseline effects, remodeled mitochondrial redox readouts consistent with controlled ROS signaling, while improving mitochondrial content/architecture and blunting stress-evoked compensatory glycolysis. MNP-021 restored pro-survival signaling (pAKT/pERK and nuclear pCREB), including insulin responsiveness during MGO exposure. MNP-021 reduced IL-6/TNF- release while increasing IL-10 and ARG1 ([~]1.9-fold vs LPS/TNF-) in HMC3 glial cells, shifting them toward a pro-resolving IL-10/ARG1 program with reduced A{beta}(25-35)-evoked cytokine release with GLP-1 remaining very low ([≤]10 pg/mL) and not significantly increased in this system. ConclusionsMNP-021 coordinates transcriptomic restraint, transporter-level glucose handling, mitochondrial resilience, and pro-survival/pro-resolving signaling across neuron-microglia compartments, supporting TRPA1-tuned small-molecule modulation as a candidate strategy against dicarbonyl-linked neuro-metabolic stress.

physiology↗

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

Simulating BRAFV600E-MEK-ERK signalling dynamics in response to vertical inhibition treatment strategies

In vertical inhibition treatment strategies, multiple components of an intracellular pathway are simulta-neously inhibited. Vertical inhibition of the BRAFV600E-MEK-ERK signalling pathway is a standard of care for treating BRAFV600E-mutated melanoma where two targeted cancer drugs, a BRAFV600E-inhibitor, and a MEK-inhibitor, are administered in combination. Targeted therapies have been linked to early onsets of drug resistance, and thus treatment strategies of higher complexities and lower doses have been proposed as alternatives to current clinical strategies. However, finding optimal complex, low-dose treatment strategies is a challenge, as it is possible to design more treatment strategies than are feasibly testable in experimental settings. To quantitatively address this challenge, we develop a mathematical model of BRAFV600E-MEK-ERK signalling dynamics in response to combinations of the BRAFV600E-inhibitor dabrafenib (DBF), the MEK-inhibitor trametinib (TMT), and the ERK-inhibitor SCH772984 (SCH). From a model of the BRAFV600E-MEK-ERK pathway, and a set of molecular-level drug-protein interactions, we extract a system of chemical reactions that is parameterized by in vitro data and converted to a system of ordinary differential equations (ODEs) using the law of mass action. The ODEs are solved numerically to produce simulations of how pathway-component concentrations change over time in response to different treatment strategies, i.e., inhibitor combinations and doses. The model can thus be used to limit the search space for effective treatment strategies that target the BRAFV600E-MEK-ERK pathway and warrant further experimental investigation. The results demonstrate that DBF and DBF-TMT-SCH therapies show marked sensitivity to BRAFV600E concentrations in silico, whilst TMT and SCH monotherapies do not.

cancer biology↗

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↗