Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

Mycobacterium tuberculosis Antigen 85B modifies BCG-induced anti-tuberculosis immunity and favors pathogen survival.

Tuberculosis is one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis (Mtb) has developed strategies not only to evade from host immunity but also to manipulate it for its survival. We investigated whether Mtb exploited the immunogenicity of Ag85B, one of its major secretory proteins, to redirect host anti-TB immunity to its advantage. We found that administration of Ag85B protein to mice vaccinated with Bacillus Calmette-Guérin (BCG) impaired the protection elicited by vaccination causing a more severe infection when mice were challenged with Mtb. Ag85B administration reduced BCG-induced CD4 T cell activation and IFN-γ, CCL-4 and IL-22 production in response to Mtb-infected cells. On the other hand, it promoted robust Ag85B-responsive IFN-γ-producing CD4 T cells, expansion of a subset of IFN-γ/IL-10-producing CD4+FOXP3+Treg cells, differential activation of IL-17/IL-22 responses and activation of regulatory and exhaustion pathways, including programmed death-ligand 1 expression on macrophages. All this resulted in impaired intracellular Mtb growth control by systemic immunity, both at pre- and post-Mtb challenge. Interestingly, Mtb infection itself generated Ag85B-reactive inflammatory immune cells incapable of clearing Mtb in both unvaccinated and BCG-vaccinated mice. Our data suggest that Mtb can exploit the strong immunogenicity of Ag85B to promote its own survival and spread. Since Ag85B is normally secreted by replicating bacteria and it is commonly found in the lungs of the Mtb-infected host, our findings may advance the understanding on the mechanisms of Mtb pathogenesis and immune evasion.

CD8+CD103+PD1+TIM3+ T cells in glioblastoma microenvironment correlate with prognosis.

Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.

Effects of a gut-selective integrin-targeted therapy in male mice exposed to early immune activation, a model for the study of autism spectrum disorder.

To clarify the role of gut mucosal immunity in ASD, we evaluated, in the early-life immune activation (EIA) mouse model, the effects of administration of a monoclonal antibody directed against the integrin alpha4 beta7 (α4ß7 mAb), blocking the leukocyte homing into the gut mucosa. EIA is a double-hit variant of the maternal immune-activation (MIA) model, including both prenatal (Poly I:C) and postnatal (LPS) immune challenges. In C57BL6/J EIA male adult offspring mice, IL-1ß and IL-17A mRNA colonic tissue content increased when compared with controls. Cytofluorimetric analyses of lymphocytes isolated from mesenteric lymph-nodes (MLN) and spleens of EIA mice show increased percentage of total and CD4+α4ß7+, unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in MLN and CD4+α4ß7+ unstimulated and stimulated IL-17A+ and stimulated IFN-γ+ lymphocytes in the spleen. Treatment with anti-α4ß7 mAb in EIA male mice was associated with colonic tissue IL-1ß, and IL-17A mRNA content and percentage of CD4+ IL-17A+ and IFN-γ+ lymphocytes in MLN and spleens comparable to control mice. The anti-α4ß7 mAb treatment rescue social novelty deficit showed in the three-chamber test by EIA male mice. Increased levels of IL-6 and IL-1ß and decreased CD68 and TGF-ß mRNAs were also observed in hippocampus and prefrontal cortex of EIA male mice together with a reduction of BDNF mRNA levels in all brain regions examined. Anti-α4ß7 mAb treatment restored the expression of BDNF, TGF-ß and CD68 in hippocampus and prefrontal cortex. Improvement of the gut inflammatory status, obtained by a pharmacological agent acting exclusively at gut level, ameliorates some ASD behavioral features and the neuroinflammatory status. Data provide the first preclinical indication for a therapeutic strategy against gut-immune activation in ASD subjects with peripheral increase of gut-derived (α4ß7+) lymphocytes expressing IL-17A.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

Extracellular DNA secreted in yeast cultures is metabolism-specific and inhibits cell proliferation.

Extracellular DNA (exDNA) can be actively released by living cells and different putative functions have been attributed to it. Further, homologous exDNA has been reported to exert species-specific inhibitory effects on several organisms. Here, we demonstrate by different experimental evidence, including 1H-NMR metabolomic fingerprint, that the growth rate decline in Saccharomyces cerevisiae fed-batch cultures is determined by the accumulation of exDNA in the medium. Sequencing of such secreted exDNA represents a portion of the entire genome, showing a great similarity with extrachromosomal circular DNA (eccDNA) already reported inside yeast cells. The recovered DNA molecules were mostly single strands and specifically associated to the yeast metabolism displayed during cell growth. Flow cytometric analysis showed that the observed growth inhibition by exDNA corresponded to an arrest in the S phase of the cell cycle. These unprecedented findings open a new scenario on the functional role of exDNA produced by living cells.

A cellular screening platform, stably expressing DENV2 NS5, defines a novel anti-DENV mechanism of action of Apigenin based on STAT2 activation.

Type I interferon (IFN-I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the "promiscuous" and "pan-assay-interfering" nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses.

Inhibition of cholesterol transport impairs Cav-1 trafficking and small extracellular vesicles secretion, promoting amphisome formation in melanoma cells.

Caveolin-1 (Cav-1) is a fundamental constituent of caveolae, whose functionality and structure are strictly dependent on cholesterol. In this work the U18666A inhibitor was used to study the role of cholesterol transport in the endosomal degradative-secretory system in a metastatic human melanoma cell line (WM266-4). We found that U18666A induces a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, which is involved, through Multi Vesicular Bodies (MVBs), in the formation and release of small extracellular vesicles (sEVs). Moreover, this inhibitor induces an increase in the production of sEVs with chemical-physical characteristics similar to control sEVs but with a different protein composition (lower expression of Cav-1 and increase of LC3II) and reduced transfer capacity on target cells. Furthermore, we determined that U18666A affects mitochondrial function and also cancer cell aggressive features, such as migration and invasion. Taken together, these results indicate that the blockage of cholesterol transport, determining the internalization of Cav-1, may modify sEVs secretory pathways through an increased fusion between autophagosomes and MVBs to form amphisome, which in turn fuses with the plasma membrane releasing a heterogeneous population of sEVs to maintain homeostasis and ensure correct cellular functionality.

Isoginkgetin-A Natural Compound to Control U87MG Glioblastoma Cell Growth and Migration Activating Apoptosis and Autophagy.

Isoginkgetin (Iso) is a natural bioflavonoid isolated from the leaves of Ginkgo biloba, this natural substance exhibits many healing properties, among which the antitumor effect stands out. Here we tested the effect of Iso on the growth of U87MG glioblastoma cells. Growth curves and MTT toxicity assays showed time and dose-dependent growth inhibition of U87MG after treatment with Iso (15/25 µM) for 1, 2, and 3 days. The cell growth block of U87MG was further investigated with the colony formation test, which showed that iso treatment for 24 h reduced colony formation. The present study also aimed to evaluate the effect of Iso on U87MG glioblastoma cell migration. The FACS analysis, on the other hand, showed that treatment with Iso 15 µM determines a blockage of the cell cycle in the S1 phase. Further investigation shows that Iso treatment of U87MG altered the protein pathways of homeostasis including autophagy and apoptosis. The present study demonstrated, for the first time, that Iso could represent an excellent adjuvant drug for the treatment of glioblastoma by simultaneously activating multiple mechanisms that control the growth and migration of neoplastic cells.

An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory.

The common view is that T lymphocytes activate telomerase to delay senescence. Here we show that some T cells (primarily naïve and central memory cells) elongated telomeres by acquiring telomere vesicles from antigen-presenting cells (APCs) independently of telomerase action. Upon contact with these T cells, APCs degraded shelterin to donate telomeres, which were cleaved by the telomere trimming factor TZAP, and then transferred in extracellular vesicles at the immunological synapse. Telomere vesicles retained the Rad51 recombination factor that enabled telomere fusion with T-cell chromosome ends lengthening them by an average of ~3,000 base pairs. Thus, there are antigen-specific populations of T cells whose ageing fate decisions are based on telomere vesicle transfer upon initial contact with APCs. These telomere-acquiring T cells are protected from senescence before clonal division begins, conferring long-lasting immune protection.

Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B.

Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I → KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.

Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein.

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

A Soft Zwitterionic Hydrogel as Potential Coating on a Polyimide Surface to Reduce Foreign Body Reaction to Intraneural Electrodes.

Invasive intraneural electrodes can control advanced neural-interfaced prostheses in human amputees. Nevertheless, in chronic implants, the progressive formation of a fibrotic capsule can gradually isolate the electrode surface from the surrounding tissue leading to loss of functionality. This is due to a nonspecific inflammatory response called foreign-body reaction (FBR). The commonly used poly(ethylene glycol) (PEG)-based low-fouling coatings of implantable devices can be easily encapsulated and are susceptible to oxidative damage in long-term in vivo applications. Recently, sulfobetaine-based zwitterionic hydrogels have emerged as an important class of robust ultra-low fouling biomaterials, holding great potential to mitigate FBR. The aim of this proof-of-principle in vitro work was to assess whether the organic zwitterionic-poly(sulfobetaine methacrylate) [poly(SBMA)]-hydrogel could be a suitable coating for Polyimide (PI)-based intraneural electrodes to reduce FBR. We first synthesized and analyzed the hydrogel through a mechanical characterization (i.e., Young's modulus). Then, we demonstrated reduced adhesion and activation of fibrogenic and pro-inflammatory cells (i.e., human myofibroblasts and macrophages) on the hydrogel compared with PEG-coated and polystyrene surfaces using cell viability assays, confocal fluorescence microscopy and high-content analysis of oxidative stress production. Interestingly, we successfully coated PI surfaces with a thin film of the hydrogel through covalent bond and demonstrated its high hydrophilicity via water contact angle measurement. Importantly, we showed the long-term release of an anti-fibrotic drug (i.e., Everolimus) from the hydrogel. Because of the low stiffness, biocompatibility, high hydration and ultra-low fouling characteristics, our zwitterionic hydrogel could be envisioned as long-term diffusion-based delivery system for slow and controlled anti-inflammatory and anti-fibrotic drug release in vivo.

Strong SARS-CoV-2 N-Specific CD8+ T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice.

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.

Inhibiting effect of p-Coumaric acid on U87MG human glioblastoma cell growth.

p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.

CD146 expression regulates osteochondrogenic differentiation of human adipose-derived stem cells.

Tissue engineering aims to develop innovative approaches to repair tissue defects. The use of adipose-derived stem cells (ASCs) in tissue regeneration was extensively investigated for osteochondrogenesis. Among the ASC population, ASCs expressing the CD146 were demonstrated to be multipotent and considered as perivascular stem cells, although the functional role of CD146 expression in these cells remains unclear. Herein, we investigated the influence of CD146 expression on osteochondrogenic differentiation of ASCs. Our results showed that, in two-dimensional culture systems, sorted CD146+ ASCs proliferated less and displayed higher adipogenic and chondrogenic potential than CD146- ASCs. The latter demonstrated a higher osteogenic capacity. Besides this, CD146+ ASCs in three-dimensional Matrigel/endothelial growth medium (EGM) cultures showed the highest angiogenic capability. When cultured in three-dimensional collagen scaffolds, CD146+ ASCs showed a spontaneous chondrogenic differentiation, further enhanced by the EGM medium's addition. Finally, CD146- ASCs seeded on hexafluoroisopropanol silk scaffolds displayed a greater spontaneous osteogenetic capacity. Altogether, these findings demonstrated a functional and relevant influence of CD146 expression in ASC properties and osteochondrogenic commitment. Exploiting the combination of specific differentiation properties of ASC subpopulations and appropriate culture systems could represent a promising strategy to improve the efficacy of new regenerative therapies.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

A cytofluorimetric analysis of a Saccharomyces cerevisiae population cultured in a fed-batch bioreactor.

The yeast Saccharomyces cerevisiae is a reference model system and one of the widely used microorganisms in many biotechnological processes. In industrial yeast applications, combined strategies aim to maximize biomass/product yield, with the fed-batch culture being one of the most frequently used. Flow cytometry (FCM) is widely applied in biotechnological processes and represents a key methodology to monitor cell population dynamics. We propose here an application of FCM in the analysis of yeast cell cycle along the time course of a typical S. cerevisiae fed-batch culture. We used two different dyes, SYTOX Green and SYBR Green, with the aim to better define each stage of cell cycle during S. cerevisiae fed-batch culture. The results provide novel insights in the use of FCM cell cycle analysis for the real-time monitoring of S. cerevisiae bioprocesses.

Enzymatically active apurinic/apyrimidinic endodeoxyribonuclease 1 is released by mammalian cells through exosomes.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.

Functional analysis of POLD1 p.ser605del variant: the aging phenotype of MDPL syndrome is associated with an impaired DNA repair capacity.

Mandibular hypoplasia, Deafness and Progeroid features with concomitant Lipodystrophy define a rare systemic disorder, named MDPL Syndrome, due to almost always a de novo variant in POLD1 gene, encoding the DNA polymerase δ. We report a MDPL female heterozygote for the recurrent p.Ser605del variant. In order to deepen the functional role of the in frame deletion affecting the polymerase catalytic site of the protein, cellular phenotype has been characterised. MDPL fibroblasts exhibit in vitro nuclear envelope anomalies, accumulation of prelamin A and presence of micronuclei. A decline of cell growth, cellular senescence and a blockage of proliferation in G0/G1 phase complete the aged cellular picture. The evaluation of the genomic instability reveals a delayed recovery from DNA induced-damage. Moreover, the rate of telomere shortening was greater in pathological cells, suggesting the telomere dysfunction as an emerging key feature in MDPL. Our results suggest an alteration in DNA replication/repair function of POLD1 as a primary pathogenetic cause of MDPL. The understanding of the mechanisms linking these cellular characteristics to the accelerated aging and to the wide spectrum of affected tissues and clinical symptoms in the MDPL patients may provide opportunities to develop therapeutic treatments for progeroid syndromes.