NG2 is the rat homologue of the human melanoma chondroitin sulfate proteoglycan (MCSP) preferentially expressed in dividing progenitor cells of the glial and mesenchymal lineage but downregulated after differentiation. It has recently been demonstrated that MCSP/NG2 expression is not restricted to mitotic or malignant cells. We show that MCSP/NG2 expression is detectable in the sarcolemma, and in the neuromuscular junction of human postnatal skeletal muscle, and it gradually reduces with advancing age. In human and murine myogenic cell lines, we found no clear differences in MCSP/NG2 expression between myoblasts and myotubes. Reduced levels of the core protein were found in merosin-negative congenital muscular dystrophy (MDC1A). Duchenne muscular dystrophy patients muscles exhibited an overexpression of the MCSP/NG2 core protein. In gamma-sarcoglycanopathy and calpainopathy, MCSP/NG2 upregulation was restricted to regenerating myofibers. We demonstrate that MCSP/NG2 is expressed in differentiated myofibers, and appears to have a role in the pathogenesis of MDC1A and severe dystrophinopathies.

Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specific recognition of RNA cis-acting signals located at the ends of the viral genome. To identify cellular proteins involved in coronavirus RNA synthesis, transmissible gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for RNA affinity protein purification. Ten proteins were preferentially pulled down with either the 5' or 3' ends of the genome and identified by proteomic analysis. Nine of them, including members of the heterogeneous ribonucleoprotein family of proteins (hnRNPs), the poly(A)-binding protein (PABP), the p100 transcriptional co-activator protein and two aminoacyl-tRNA synthetases, showed a preferential binding to the 3' end of the genome, whereas only the polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5' end of the genome. The potential function of the 3' end-interacting proteins in virus replication was studied by analyzing the effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP, hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) caused a significant 2 to 3-fold reduction of viral RNA synthesis. Interestingly, the silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in viral RNA synthesis in both systems. These data suggest that PABP, hnRNP Q, and EPRS play a positive role in virus infection that could be mediated through their interaction with the viral 3' end, and that GAPDH has a negative effect on viral infection.

The large size of the coronavirus (CoV) genome (around 30 kb) and the instability in bacteria of plasmids carrying CoV replicase sequences represent serious restrictions for the development of CoV infectious clones using reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these problems, several approaches have been established in the last 13 years. Here we describe the engineering of CoV full-length cDNA clones as bacterial artificial chromosomes (BACs), using the Middle East respiratory syndrome CoV (MERS-CoV) as a model.

Atypical fibroblast growth factors (FGF) 21 and 19 play a central role in energy metabolism through the mediation of Klotho coreceptor. Contradictory findings are available about the association of FGF21 and FGF19 with nonalcoholic fatty liver disease (NAFLD) in humans. We investigated the association of serum FGF21, FGF19 and liver Klotho coreceptor with non-alcoholic steatohepatitis (NASH) and fibrosis in children with NAFLD. Serum FGF21 and FGF19 were measured in 84 children with biopsy-proven NAFLD and 23 controls (CTRL). The hepatic expression of Klotho coreceptor was measured in 7 CTRL, 9 patients with NASH (NASH+) and 11 patients without NASH (NASH-). FGF21 and FGF19 showed a tendency to decrease from CTRL (median FGF21 = 196 pg/mL; median FGF19 = 201 pg/mL) to NASH- (FGF21 = 89 pg/mL; FGF19 = 81 pg/mL) to NASH+ patients (FGF21 = 54 pg/mL; FGF19 = 41 pg/mL) (p<0.001 for all comparisons) and were inversely associated with the probability of NASH and fibrosis in children with NAFLD. The hepatic expression of Klotho coreceptor was inversely associated with NASH (R(2) = 0.87, p<0.0001) and directly associated with serum FGF21 (R(2) = 0.57, p<0.0001) and FGF19 (R(2) = 0.67, p<0.0001). In conclusion, serum FGF19 and FGF21 and hepatic Klotho expression are inversely associated with hepatic damage in children with NAFLD and these findings may have important implications for understanding the mechanisms of NAFLD progression.

Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A 'One Health' vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs.

Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus.

Mammalian tissues display a remarkable complexity of splicing patterns. Nevertheless, only few examples of tissue-specific splicing regulators are known. Herein, we characterize a novel splicing regulator named RBM11, which contains an RNA Recognition Motif (RRM) at the amino terminus and a region lacking known homology at the carboxyl terminus. RBM11 is selectively expressed in brain, cerebellum and testis, and to a lower extent in kidney. RBM11 mRNA levels fluctuate in a developmentally regulated manner, peaking perinatally in brain and cerebellum, and at puberty in testis, in concomitance with differentiation events occurring in neurons and germ cells. Deletion analysis indicated that the RRM of RBM11 is required for RNA binding, whereas the carboxyl terminal region permits nuclear localization and homodimerization. RBM11 is localized in the nucleoplasm and enriched in SRSF2-containing splicing speckles. Transcription inhibition/release experiments and exposure of cells to stress revealed a dynamic movement of RBM11 between nucleoplasm and speckles, suggesting that its localization is affected by the transcriptional status of the cell. Splicing assays revealed a role for RBM11 in the modulation of alternative splicing. In particular, RBM11 affected the choice of alternative 5' splice sites in BCL-X by binding to specific sequences in exon 2 and antagonizing the SR protein SRSF1. Thus, our findings identify RBM11 as a novel tissue-specific splicing factor with potential implication in the regulation of alternative splicing during neuron and germ cell differentiation.

NG2, the rat homologue of the human melanoma chondroitin sulfate proteoglycan (MCSP), is a ligand for collagen VI (COL6). We have examined skeletal muscles of patients affected by Ullrich scleroatonic muscular dystrophy (UCMD), an inherited syndrome caused by COL6 genes mutations. A significant decrease of NG2 immunolabeling was found in UCMD myofibers, as well as in skeletal muscle and cornea of COL6 null-mice. In UCMD muscles, truncated NG2 core protein isoforms were detected. However, real-time RT-PCR analysis revealed marked increase in NG2 mRNA content in UCMD muscle compared to controls. We hypothesize that NG2 immunohistochemical and biochemical behavior may be compromised owing to the absence of its physiological ligand. MCSP/NG2 proteoglycan may be considered an important receptor mediating COL6-sarcolemma interactions, a relationship that is disrupted by the pathogenesis of UCMD muscle.

Under physiological conditions, PD-1/PD-L1 interactions regulate unwanted over-reactions of immune cells and contribute to maintain peripheral tolerance. However, in tumor microenvironment, this interaction may greatly compromise the immune-mediated anti-tumor activity. PD-1+ NK cells have been detected in high percentage in peripheral blood and ascitic fluid of ovarian carcinoma patients. To acquire information on PD-1 expression and physiology in human NK cells, we analyzed whether PD-1 mRNA and protein are present in resting, surface PD-1-, NK cells from healthy donors. Both different splicing isoforms of PD-1 mRNA and a cytoplasmic pool of PD-1 protein were detected. Similar results were obtained also from both in vitro-activated and tumor-associated NK cells. PD-1 mRNA and protein were higher in CD56dim than in CD56bright NK cells. Confocal microscopy analyses revealed that PD-1 protein is present in virtually all NK cells analyzed. The present findings are compatible with a rapid surface expression of PD-1 in NK cells in response to appropriate, still undefined, stimuli.

Influenza's propensity for antigenic drift and shift, and to elicit predominantly strain specific antibodies (Abs) leaves humanity susceptible to waves of new strains with pandemic potential for which limited or no immunity may exist. Subsequently new clinical interventions are needed. To identify hemagglutinin (HA) epitopes that if targeted may confer universally protective humoral immunity, we examined plasmablasts from a subject that was immunized with the seasonal influenza inactivated vaccine, and isolated a human monoclonal Ab (mAb), KPF1. KPF1 has broad and potent neutralizing activity against H1 influenza viruses, and recognized 83% of all H1 isolates tested, including the pandemic 1918 H1. Prophylactically, KPF1 treatment resulted in 100% survival of mice from lethal challenge with multiple H1 influenza strains and when given as late as 72 h after challenge with A/California/04/2009 H1N1, resulted in 80% survival. KPF1 recognizes a novel epitope in the HA globular head, which includes a highly conserved amino acid, between the Ca and Cb antigenic sites. Although recent HA stalk-specific mAbs have broader reactivity, their potency is substantially limited, suggesting that cocktails of broadly reactive and highly potent HA globular head-specific mAbs, like KPF1, may have greater clinical feasibility for the treatment of influenza infections.

Numerous infectious diseases impacting livestock impose an important economic burden and in some cases also represent a threat to humans and are classified as zoonoses. Some zoonotic diseases are transmitted by vectors and, due to complex environmental and socio-economic factors, the distribution of many of these pathogens is changing, with increasing numbers being found in previously unaffected countries. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies to five important pathogens of livestock (three of them zoonotic) that are currently emerging in new geographical locations: Rift Valley fever virus (RVFV), Crimean-Congo haemorrhagic fever virus (CCHFV), Schmallenberg virus (SBV), Bluetongue virus (BTV) and the bacteria complex Mycobacterium tuberculosis. Using the Luminex platform, polystyrene microspheres were coated with recombinant proteins from each of the five pathogens. The mix of microspheres was used for the simultaneous detection of antibodies against the five corresponding diseases affecting ruminants. The following panel of sera was included in the study: 50 sera from sheep experimentally infected with RVFV, 74 sera from calves and lambs vaccinated with SBV, 26 sera from cattle vaccinated with Mycobacterium bovis, 30 field sera from different species of ruminants infected with CCHFV and 88 calf sera infected with BTV. Finally, to determine its diagnostic specificity 220 field sera from Spanish farms free of the five diseases were assessed. All the sera were classified using commercial ELISAs specific for each disease, used in this study as the reference technique. The results showed the multiplex assay exhibited good performance characteristics with values of sensitivity ranging from 93% to 100% and of specificity ranging from 96% to 99% depending on the pathogen. This new tool allows the simultaneous detection of antibodies against five important pathogens, reducing the volume of sample needed and the time of analysis where these pathogens are usually tested individually.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Although it is well established that Influenza A virus infection is initiated in the respiratory tract, the sequence of events and the cell types that become infected or access viral antigens remains incompletely understood. In this report, we used a novel Influenza A/California/04/09 (H1N1) reporter virus that stably expresses the Venus fluorescent protein to identify antigen-bearing cells over time in a mouse model of infection using flow cytometry. These studies revealed that many hematopoietic cells, including subsets of monocytes, macrophages, dendritic cells, neutrophils and eosinophils acquire influenza antigen in the lungs early post-infection. Surface staining of the viral HA revealed that most cell populations become infected, most prominently CD45neg cells, alveolar macrophages and neutrophils. Finally, differences in infection status, cell lineage and MHC class II expression by antigen-bearing cells correlated with differences in their ability to re-stimulate influenza-specific CD4 T cells ex vivo. Collectively, these studies have revealed the cellular heterogeneity and complexity of antigen-bearing cells within the lung and their potential as targets of antigen recognition by CD4 T cells.

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown-Vialetto-Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell-selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy-lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

Schmallenberg virus (SBV), an arthropod-transmitted pathogenic bunyavirus, continues to be a threat to the European livestock industry, causing morbidity and mortality among young ruminant livestock. Here, we describe a novel SBV subunit vaccine, based on bacterially expressed SBV nucleoprotein (SBV-N) administered with a veterinary-grade Saponin adjuvant. When assayed in an IFNAR-/- mouse model, SBV-N with Saponin induced strong non-neutralizing broadly virus-reactive antibodies, decreased clinical signs, as well as significantly reduced viremia. Vaccination assays also suggest that this level of immune protection is cell mediated, as evidenced by the lack of neutralizing antibodies, as well as interferon-γ secretion observed in vitro. Therefore, based on these results, bacterially expressed SBV-N, co-administered with veterinary-grade Saponin adjuvant may serve as a promising economical alternative to current SBV vaccines, and warrant further evaluation in large ruminant animal models. Moreover, we propose that this strategy may be applicable to other bunyaviruses.

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift. Previously, we described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV) in vitro, and prophylactic and therapeutic activities in vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrated how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian-produced KPF1-HEK hMAb. KPF1-Antx hMAb showed broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, which was comparable to that observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrated, for the first time, a plant-produced influenza hMAb with in vitro and in vivo activity against influenza virus. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.

Chimeric antigen receptor (CAR) T-cell therapy has been shown to be dramatically effective in the treatment of B-cell malignancies. However, there are still substantial obstacles to overcome, before similar responses can be achieved in patients with solid tumors. We evaluated both in vitro and in a preclinical murine model the efficacy of different 2nd and 3rd generation CAR constructs targeting GD2, a disial-ganglioside expressed on the surface of neuroblastoma (NB) tumor cells. In order to address potential safety concerns regarding clinical application, an inducible safety switch, namely inducible Caspase-9 (iC9), was also included in the vector constructs. Our data indicate that a 3rd generation CAR incorporating CD28.4-1BB costimulatory domains is associated with improved anti-tumor efficacy as compared with a CAR incorporating the combination of CD28.OX40 domains. We demonstrate that the choice of 4-1BB signaling results into significant amelioration of several CAR T-cell characteristics, including: 1) T-cell exhaustion, 2) basal T-cell activation, 3) in vivo tumor control and 4) T-cell persistence. The fine-tuning of T-cell culture conditions obtained using IL7 and IL15 was found to be synergic with the CAR.GD2 design in increasing the anti-tumor activity of CAR T cells. We also demonstrate that activation of the suicide gene iC9, included in our construct without significantly impairing neither CAR expression nor anti-tumor activity, leads to a prompt induction of apoptosis of GD2.CAR T cells. Altogether, these findings are instrumental in optimizing the function of CAR T-cell products to be employed in the treatment of children with NB.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore, it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an interferon (IFN)-competent cell line as well as the production of interferon beta (IFN-β) did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results highlight the key role of the NSs protein in the modulation of viral infectivity.

Interferons (IFNs), IFN-stimulated genes (ISGs), and inflammatory cytokines mediate innate immune responses, and are essential to establish an antiviral response. Within the innate immune responses, retinoic acid-inducible gene I (RIG-I) is a key sensor of virus infections, mediating the transcriptional induction of IFNs and inflammatory proteins. Nevertheless, since excessive responses could be detrimental to the host, these responses need to be tightly regulated. In this work, we describe, for the first time, how knocking-down or knocking-out the expression of IFN alpha-inducible protein 6 (IFI6) increases IFN, ISG, and pro-inflammatory cytokine expression after the infections with Influenza A Virus (IAV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and Sendai Virus (SeV), or poly(I:C) transfection. We also show how overexpression of IFI6 produces the opposite effect, in vitro and in vivo, indicating that IFI6 negatively modulates the induction of innate immune responses. Knocking-out or knocking-down the expression of IFI6 diminishes the production of infectious IAV and SARS-CoV-2, most likely because of its effect on antiviral responses. Importantly, we report a novel interaction of IFI6 with RIG-I, most likely mediated through binding to RNA, that affects RIG-I activation, providing a molecular mechanism for the effect of IFI6 on negatively regulating innate immunity. Remarkably, these new functions of IFI6 could be targeted to treat diseases associated with an exacerbated induction of innate immune responses and to combat viral infections, such as IAV and SARS-CoV-2.

The existence of individuals who remain cognitively intact despite presenting histopathological signs of Alzheimer's disease (AD), here referred to as "Nondemented with AD neuropathology" (NDAN), suggests that some mechanisms are triggered to resist cognitive impairment. Exposed phosphatidylserine (ePS) represents a neuronal "eat-me" signal involved in microglial-mediated phagocytosis of damaged synapses. A possible mediator of this process is TREM2, a microglial surface receptor activated by ligands including PS. Based on TREM2 role in the scavenging function of microglia, we hypothesize that an efficient microglial phagocytosis of damaged synapses underlies synaptic resilience in NDAN, thus protecting from memory deficits. Using immunofluorescence microscopy, we performed a comparative study of human post-mortem frontal cortices of aged-matched, AD and NDAN individuals. We studied the distribution of activated microglia (IBA1, IBA1+ /CD68+ cells) and phagocytic microglia-related proteins (TREM2, DAP12), demonstrating higher microglial activation and TREM2 expression in NDAN versus AD. A study of the preservation of synapses around plaques, assessed using MAP2 and βIII tubulin as dendritic and axonal markers, respectively, and PSD95 as a postsynaptic marker, revealed preserved axonal/dendritic structure around plaques in NDAN versus AD. Moreover, high levels of PSD95 around NDAN plaques and the colocalization of PSD95 with CD68 indicated a prompt removal of damaged synapses by phagocytic microglia. Furthermore, Annexin V assay on aged-matched, AD and NDAN individuals synaptosomes revealed increased levels of ePS in NDAN, confirming damaged synapses engulfment. Our results suggest a higher efficiency of TREM2-induced phagocytic microglia in removing damaged synapses, underlying synaptic resilience in NDAN individuals.