Extracellular vesicles released by microglia and macrophages carry endocannabinoids which foster oligodendrocyte differentiation.

Introduction: Microglia and macrophages can influence the evolution of myelin lesions through the production of extracellular vesicles (EVs). While microglial EVs promote in vitro differentiation of oligodendrocyte precursor cells (OPCs), whether EVs derived from macrophages aid or limit OPC maturation is unknown. Methods: Immunofluorescence analysis for the myelin protein MBP was employed to evaluate the impact of EVs from primary rat macrophages on cultured OPC differentiation. Raman spectroscopy and liquid chromatography-mass spectrometry was used to define the promyelinating lipid components of myelin EVs obtained in vitro and isolated from human plasma. Results and discussion: Here we show that macrophage-derived EVs do not promote OPC differentiation, and those released from macrophages polarized towards an inflammatory state inhibit OPC maturation. However, their lipid cargo promotes OPC maturation in a similar manner to microglial EVs. We identify the promyelinating endocannabinoids anandamide and 2-arachidonoylglycerol in EVs released by both macrophages and microglia in vitro and circulating in human plasma. Analysis of OPC differentiation in the presence of the endocannabinoid receptor antagonists SR141716A and AM630 reveals a key role of vesicular endocannabinoids in OPC maturation. From this study, EV-associated endocannabinoids emerge as important mediators in microglia/macrophage-oligodendrocyte crosstalk, which may be exploited to enhance myelin repair.

PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ.

Strigolactones as Broad-Spectrum Antivirals against ß-Coronaviruses through Targeting the Main Protease Mpro.

The current SARS-CoV-2 pandemic and the likelihood that new coronavirus strains will emerge in the immediate future point out the urgent need to identify new pan-coronavirus inhibitors. Strigolactones (SLs) are a class of plant hormones with multifaceted activities whose roles in plant-related fields have been extensively explored. Recently, we proved that SLs also exert antiviral activity toward herpesviruses, such as human cytomegalovirus (HCMV). Here we show that the synthetic SLs TH-EGO and EDOT-EGO impair ß-coronavirus replication including SARS-CoV-2 and the common cold human coronavirus HCoV-OC43. Interestingly, in silico simulations suggest the binding of SLs in the SARS-CoV-2 main protease (Mpro) active site, and this was further confirmed by an in vitro activity assay. Overall, our results highlight the potential efficacy of SLs as broad-spectrum antivirals against ß-coronaviruses, which may provide the rationale for repurposing this class of hormones for the treatment of COVID-19 patients.

One-Year Real-World Study on Comparison among Different Continuous Subcutaneous Insulin Infusion Devices for the Management of Pediatric Patients with Type 1 Diabetes: The Supremacy of Hybrid Closed-Loop Systems.

Since their advent in daily clinical practice, continuous subcutaneous insulin infusion (CSII) systems have been increasingly improved, leading to a high percentage of both adult and pediatric patients with diabetes now using insulin pumps. Different types of CSII systems are currently available, which are characterized by different settings and technical features. This longitudinal observational study aims to evaluate real-word glycemic outcomes in children and adolescents with type 1 diabetes using three different CSII devices: hybrid closed-loop (HCL) systems, predictive low glucose (PLGS) systems, and non-automated insulin pumps. The secondary objective was to identify clinical variables that may significantly influence the achievement of therapeutic goals in our study cohort. One-hundred-and-one patients on CSII therapy attending our pediatric diabetes center were enrolled. When compared with the non-automated group, patients using HCL systems showed higher levels of time in target glucose range (p = 0.003) and lower glucose variability (p = 0.008). Similarly, we found significantly better glucose metrics in HCL users in comparison to PLGS patients (time in range p = 0.008; coefficient of variation p = 0.009; time above 250 mg/dL p = 0.007). Multiple linear regression models showed that HCL systems (time in range p < 0.001) and high daily percentage of glycemic sensor use (time in range p = 0.031) are predictors for good glycemic control. The introduction and increasing availability of novel technologies for diabetes represent a promising strategy to improve glycemic control and quality of life in pediatric patients with type 1 diabetes. Our real-world data confirm the superiority of HCL systems in terms of improvement of time spent in the target glucose range, prevention of hypoglycemia, and reduction of glycemic variability.

Exploring Ligand Binding Domain Dynamics in the NRs Superfamily.

Nuclear receptors (NRs) are transcription factors that play an important role in multiple diseases, such as cancer, inflammation, and metabolic disorders. They share a common structural organization composed of five domains, of which the ligand-binding domain (LBD) can adopt different conformations in response to substrate, agonist, and antagonist binding, leading to distinct transcription effects. A key feature of NRs is, indeed, their intrinsic dynamics that make them a challenging target in drug discovery. This work aims to provide a meaningful investigation of NR structural variability to outline a dynamic profile for each of them. To do that, we propose a methodology based on the computation and comparison of protein cavities among the crystallographic structures of NR LBDs. First, pockets were detected with the FLAPsite algorithm and then an "all against all" approach was applied by comparing each pair of pockets within the same sub-family on the basis of their similarity score. The analysis concerned all the detectable cavities in NRs, with particular attention paid to the active site pockets. This approach can guide the investigation of NR intrinsic dynamics, the selection of reference structures to be used in drug design and the easy identification of alternative binding sites.

Microglial large extracellular vesicles propagate early synaptic dysfunction in Alzheimer's disease.

Synaptic dysfunction is an early mechanism in Alzheimer's disease that involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. Here we show that amyloid-ß released by microglia in association with large extracellular vesicles (Aß-EVs) alters dendritic spine morphology in vitro, at the site of neuron interaction, and impairs synaptic plasticity both in vitro and in vivo in the entorhinal cortex-dentate gyrus circuitry. One hour after Aß-EV injection into the mouse entorhinal cortex, long-term potentiation was impaired in the entorhinal cortex but not in the dentate gyrus, its main target region, while 24 h later it was also impaired in the dentate gyrus, revealing a spreading of long-term potentiation deficit between the two regions. Similar results were obtained upon injection of extracellular vesicles carrying Aß naturally secreted by CHO7PA2 cells, while neither Aß42 alone nor inflammatory extracellular vesicles devoid of Aß were able to propagate long-term potentiation impairment. Using optical tweezers combined to time-lapse imaging to study Aß-EV-neuron interaction, we show that Aß-EVs move anterogradely at the axon surface and that their motion can be blocked through annexin-V coating. Importantly, when Aß-EV motility was inhibited, no propagation of long-term potentiation deficit occurred along the entorhinal-hippocampal circuit, implicating large extracellular vesicle motion at the neuron surface in the spreading of long-term potentiation impairment. Our data indicate the involvement of large microglial extracellular vesicles in the rise and propagation of early synaptic dysfunction in Alzheimer's disease and suggest a new mechanism controlling the diffusion of large extracellular vesicles and their pathogenic signals in the brain parenchyma, paving the way for novel therapeutic strategies to delay the disease.

Therapeutic induction of energy metabolism reduces neural tissue damage and increases microglia activation in severe spinal cord injury.

Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged tissue suffers from a severe metabolic impairment, which aggravates axonal degeneration and neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring the spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and ROS defense which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase (ChAT)-expressing motor neurons and increased neurofilament-positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated microglia expressing the CD206 marker suggestive of a pro-resolutive, M2-like phenotype. These molecular and histological modifications observed in treated animals ultimately led to a significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism can represent an effective strategy to treat SCI.

Cooperativity as quantification and optimization paradigm for nuclear receptor modulators.

Nuclear Receptors (NRs) are highly relevant drug targets, for which small molecule modulation goes beyond a simple ligand/receptor interaction. NR-ligands modulate Protein-Protein Interactions (PPIs) with coregulator proteins. Here we bring forward a cooperativity mechanism for small molecule modulation of NR PPIs, using the Peroxisome Proliferator Activated Receptor γ (PPARγ), which describes NR-ligands as allosteric molecular glues. The cooperativity framework uses a thermodynamic model based on three-body binding events, to dissect and quantify reciprocal effects of NR-coregulator binding (K I D) and NR-ligand binding (K II D), jointly recapitulated in the cooperativity factor (α) for each specific ternary ligand·NR·coregulator complex formation. These fundamental thermodynamic parameters allow for a conceptually new way of thinking about structure-activity-relationships for NR-ligands and can steer NR modulator discovery and optimization via a completely novel approach.

A structural homologue of the plant receptor D14 mediates responses to strigolactones in the fungal phytopathogen Cryphonectria parasitica.

Strigolactones (SLs) are plant hormones and important signalling molecules required to promote arbuscular mycorrhizal (AM) symbiosis. While in plants an α/ß-hydrolase, DWARF14 (D14), was shown to act as a receptor that binds and cleaves SLs, the fungal receptor for SLs is unknown. Since AM fungi are currently not genetically tractable, in this study, we used the fungal pathogen Cryphonectria parasitica, for which gene deletion protocols exist, as a model, as we have previously shown that it responds to SLs. By means of computational, biochemical and genetic analyses, we identified a D14 structural homologue, CpD14. Molecular homology modelling and docking support the prediction that CpD14 interacts with and hydrolyses SLs. The recombinant CpD14 protein shows α/ß hydrolytic activity in vitro against the SLs synthetic analogue GR24; its enzymatic activity requires an intact Ser/His/Asp catalytic triad. CpD14 expression in the d14-1 loss-of-function Arabidopsis thaliana line did not rescue the plant mutant phenotype. However, gene inactivation by knockout homologous recombination reduced fungal sensitivity to SLs. These results indicate that CpD14 is involved in SLs responses in C. parasitica and strengthen the role of SLs as multifunctional molecules acting in plant-microbe interactions.

Plasma Small Extracellular Vesicles with Complement Alterations in GRN/C9orf72 and Sporadic Frontotemporal Lobar Degeneration.

Cutting-edge research suggests endosomal/immune dysregulation in GRN/C9orf72-associated frontotemporal lobar degeneration (FTLD). In this retrospective study, we investigated plasma small extracellular vesicles (sEVs) and complement proteins in 172 subjects (40 Sporadic FTLD, 40 Intermediate/Pathological C9orf72 expansion carriers, and 49 Heterozygous/Homozygous GRN mutation carriers, 43 controls). Plasma sEVs (concentration, size) were analyzed by nanoparticle tracking analysis; plasma and sEVs C1q, C4, C3 proteins were quantified by multiplex assay. We demonstrated that genetic/sporadic FTLD share lower sEV concentrations and higher sEV sizes. The diagnostic performance of the two most predictive variables (sEV concentration/size ratio) was high (AUC = 0.91, sensitivity 85.3%, specificity 81.4%). C1q, C4, and C3 cargo per sEV is increased in genetic and sporadic FTLD. C4 (cargo per sEV, total sEV concentration) is increased in Sporadic FTLD and reduced in GRN+ Homozygous, suggesting its specific unbalance compared with Heterozygous cases. C3 plasma level was increased in genetic vs. sporadic FTLD. Looking at complement protein compartmentalization, in control subjects, the C3 and C4 sEV concentrations were roughly half that in respect to those measured in plasma; interestingly, this compartmentalization was altered in different ways in patients. These results suggest sEVs and complement proteins as potential therapeutic targets to mitigate neurodegeneration in FTLD.

Astrocytes-derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein.

Astrocytes-derived extracellular vesicles (EVs) are key players in glia-neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV-neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)-coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin-mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface.

Binding of Androgen- and Estrogen-Like Flavonoids to Their Cognate (Non)Nuclear Receptors: A Comparison by Computational Prediction.

Flavonoids are plant bioactives that are recognized as hormone-like polyphenols because of their similarity to the endogenous sex steroids 17ß-estradiol and testosterone, and to their estrogen- and androgen-like activity. Most efforts to verify flavonoid binding to nuclear receptors (NRs) and explain their action have been focused on ERα, while less attention has been paid to other nuclear and non-nuclear membrane androgen and estrogen receptors. Here, we investigate six flavonoids (apigenin, genistein, luteolin, naringenin, quercetin, and resveratrol) that are widely present in fruits and vegetables, and often used as replacement therapy in menopause. We performed comparative computational docking simulations to predict their capability of binding nuclear receptors ERα, ERß, ERRß, ERRγ, androgen receptor (AR), and its variant ART877A and membrane receptors for androgens, i.e., ZIP9, GPRC6A, OXER1, TRPM8, and estrogens, i.e., G Protein-Coupled Estrogen Receptor (GPER). In agreement with data reported in literature, our results suggest that these flavonoids show a relevant degree of complementarity with both estrogen and androgen NR binding sites, likely triggering genomic-mediated effects. It is noteworthy that reliable protein-ligand complexes and estimated interaction energies were also obtained for some suggested estrogen and androgen membrane receptors, indicating that flavonoids could also exert non-genomic actions. Further investigations are needed to clarify flavonoid multiple genomic and non-genomic effects. Caution in their administration could be necessary, until the safe assumption of these natural molecules that are largely present in food is assured.

Can We Exploit ß-Lactamases Intrinsic Dynamics for Designing More Effective Inhibitors?

ß-lactamases (BLs) represent the most frequent cause of antimicrobial resistance in Gram-negative bacteria. Despite the continuous efforts in the development of BL inhibitors (BLIs), new BLs able to hydrolyze the last developed antibiotics rapidly emerge. Moreover, the insurgence rate of effective mutations is far higher than the release of BLIs able to counteract them. This results in a shortage of antibiotics that is menacing the effective treating of infectious diseases. The situation is made even worse by the co-expression in bacteria of BLs with different mechanisms and hydrolysis spectra, and by the lack of inhibitors able to hit them all. Differently from other targets, BL flexibility has not been deeply exploited for drug design, possibly because of the small protein size, for their apparent rigidity and their high fold conservation. In this mini-review, we discuss the evidence for BL binding site dynamics being crucial for catalytic efficiency, mutation effect, and for the design of new inhibitors. Then, we report on identified allosteric sites in BLs and on possible allosteric inhibitors, as a strategy to overcome the frequent occurrence of mutations in BLs and the difficulty of competing efficaciously with substrates. Nevertheless, allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility towards available antibiotics.

Strigolactone Analogs Are Promising Antiviral Agents for the Treatment of Human Cytomegalovirus Infection.

The human cytomegalovirus (HCMV) is a widespread pathogen and is associated with severe diseases in immunocompromised individuals. Moreover, HCMV infection is the most frequent cause of congenital malformation in developed countries. Although nucleoside analogs have been successfully employed against HCMV, their use is hampered by the occurrence of serious side effects. There is thus an urgent clinical need for less toxic, but highly effective, antiviral drugs. Strigolactones (SLs) are a novel class of plant hormones with a multifaceted activity. While their role in plant-related fields has been extensively explored, their effects on human cells and their potential applications in medicine are far from being fully exploited. In particular, their antiviral activity has never been investigated. In the present study, a panel of SL analogs has been assessed for antiviral activity against HCMV. We demonstrate that TH-EGO and EDOT-EGO significantly inhibit HCMV replication in vitro, impairing late protein expression. Moreover, we show that the SL-dependent induction of apoptosis in HCMV-infected cells is a contributing mechanism to SL antiviral properties. Overall, our results indicate that SLs may be a promising alternative to nucleoside analogs for the treatment of HCMV infections.

Multiple catalytic activities of human 17ß-hydroxysteroid dehydrogenase type 7 respond differently to inhibitors.

Cholesterol biosynthesis is a multistep process in mammals that includes the aerobic removal of three methyl groups from the intermediate lanosterol, one from position 14 and two from position 4. During the demethylations at position 4, a 3-ketosteroid reductase catalyses the conversion of both 4-methylzymosterone and zymosterone to 4-methylzymosterol and zymosterol, respectively, restoring the alcoholic function of lanosterol, which is also maintained in cholesterol. Unlike other eukaryotes, mammals also use the same enzyme as an estrone reductase that can transform estrone (E1) into estradiol (E2). This enzyme, named 17ß-hydroxysteroid dehydrogenase type 7 (HSD17B7), is therefore a multifunctional protein in mammals, and one that belongs to both the HSD17B family, which is involved in steroid-hormone metabolism, and to the family of post-squalene cholesterol biosynthesis enzymes. In the present study, a series of known inhibitors of human HSD17B7's E1-reductase activity have been assayed for potential inhibition against 3-ketosteroid reductase activity. Surprisingly, the assayed compounds lost their inhibition activity when tested in HepG2 cells that were incubated with radiolabelled acetate and against the recombinant overexpressed human enzyme incubated with 4-methylzymosterone (both radiolabelled and not). Preliminary kinetic analyses suggest a mixed or non-competitive inhibition on the E1-reductase activity, which is in agreement with Molecular Dynamics simulations. These results raise questions about the mechanism(s) of action of these possible inhibitors, the enzyme dynamic regulation and the interplay between the two activities.

Discovering New Casein Kinase 1d Inhibitors with an Innovative Molecular Dynamics Enabled Virtual Screening Workflow.

The value of including protein flexibility in structure-based drug design (SBDD) is widely documented, and currently, molecular dynamics (MD) simulations represent a powerful tool to investigate protein dynamics. Yet, the inclusion of MD-derived information in pre-existing SBDD workflows is still far from trivial. We recently published an integrated MD-FLAP (Fingerprints for Ligands and Proteins) approach combining MD, clustering and Linear Discriminant Analysis (LDA) for enhancing accuracy, efficacy, and for protein conformational selection in virtual screening (VS) campaigns. Here we prospectively applied the MD-FLAP workflow to discover novel chemotypes inhibiting the Casein Kinase 1 delta (CSNK1D) enzyme. We first obtained a VS model able to separate active from inactive compounds, with a global AUC of 0.9 and a partial ROC enrichment at 0.5% of 0.18, and use it to mine the internal Pfizer screening database. Seven active molecules sharing a phenyl-indazole scaffold, not yet reported among CSNK1D inhibitors, were found. The most potent inhibitor showed an IC50 of 134 nM.

First virtual screening and experimental validation of inhibitors targeting GES-5 carbapenemase.

The worldwide spread of beta-lactamases with hydrolytic activity extended to last resort carbapenems is aggravating the antibiotic resistance problem and endangers the successful antimicrobial treatment of clinically relevant pathogens. As recently highlighted by the World Health Organization, new strategies to contain antimicrobial resistance are urgently needed. Class A carbapenemases include members of the KPC, GES and SFC families. These enzymes have the ability to hydrolyse penicillins, cephalosporins and carbapenems, while also being less susceptible to available beta-lactam inhibitors, such as clavulanic acid. The KPC family is the most prevalent. It is mostly found on plasmids in Klebsiella pneumoniae, meaning that great amounts of attention, in terms of inhibitor design and structural biology, have been dedicated to it, whereas no efforts have yet been dedicated to GES-type enzymes, despite their ability to rapidly and horizontally disseminate. We herein report the first in silico screening against GES-5, which is the most dangerous GES-type beta-lactamase, using a library of 800K commercially available candidates that all share drug-like properties, such as their MW, logP, rotatable bonds and HBA/HBD atoms. The best screening results were filtered to enrich the number of different chemotypes, and then submitted to molecular docking. The 34 most promising candidates were selected for in vitro validation in biochemical assays against recombinant GES-5. Six hits acted as inhibitors, in the high micromolar range, towards GES-5 and led to the identification of the first, novel chemotypes with inhibitory activity against this clinically relevant carbapenemase.

Effect of fingolimod action on the release of monocyte-derived microvesicles in multiple sclerosis patients.

Recently, microvesicles (MVs) were considered as important mediators of intercellular communication, especially in pathological conditions as Multiple Sclerosis (MS). In myeloid cells, MV shedding is induced by the receptor P2X7 with the involvement of acid sphingomyelinase (A-SMase) and release of the IL-1ß. In this study we evaluate how Fingolimod affects MVs production by the monocytes, as well as P2X7R, IL-1ß expression and A-SMase activity. Treatment decreased MVs production and IL-1ß expression. This effect was associated with the inhibition of A-SMase activity in BzATP-stimulated monocytes from MS patients. These evidences suggest monocyte MVs as a possible disease and drug-efficacy biomarkers.

Glia-to-neuron transfer of miRNAs via extracellular vesicles: a new mechanism underlying inflammation-induced synaptic alterations.

Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV-neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.

Synthesis, Structure Characterization, and Evaluation in Microglia Cultures of Neuromelanin Analogues Suitable for Modeling Parkinson's Disease.

In the substantia nigra of human brain, neuromelanin (NM) released by degenerating neurons can activate microglia with consequent neurodegeneration, typical of Parkinson's disease (PD). Synthetic analogues of NM were prepared to develop a PD model reproducing the neuropathological conditions of the disease. Soluble melanin-protein conjugates were obtained by melanization of fibrillated ß-lactoglobulin (fLG). The melanic portion of the conjugates contains either eumelanic (EufLG) or mixed eumelanic/pheomelanic composition (PheofLG), the latter better simulating natural NMs. In addition, the conjugates can be loaded with controlled amounts of iron. Upon melanization, PheofLG-Fe conjugates maintain the amyloid cross-ß protein core as the only structurally organized element, similarly to human NMs. The similarity in composition and structural organization with the natural pigment is reflected by the ability of synthetic NMs to activate microglia, showing potential of the novel conjugates to model NM induced neuroinflammation. Thus, synthetic NM/microglia constitute a new model to develop anti-Parkinson drugs.