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Autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA receptors have been identified in 20%-25% of patients with frontotemporal lobar degeneration (FTLD). Data from patients and in vitro/ex vivo pre-clinical studies indicate that anti-GluA3 hIgGs negatively affect glutamatergic neurotransmission. However, whether and how the chronic presence of anti-GluA3 hIgGs triggers synaptic dysfunctions and the appearance of FTLD-related neuropathological and behavioural signature has not been clarified yet. To address this question, we developed and characterized a pre-clinical mouse model of passive immunization with anti-GluA3 hIgGs purified from patients. In parallel, we clinically compared FTLD patients who were positive for anti-GluA3 hIgGs to negative ones. Clinical data showed that the presence of anti-GluA3 hIgGs defined a subgroup of patients with distinct clinical features. In the preclinical model, anti-GluA3 hIgGs administration led to accumulation of phospho-tau in the postsynaptic fraction and dendritic spine loss in the prefrontal cortex. Remarkably, the preclinical model exhibited behavioural disturbances that mostly reflected the deficits proper of patients positive for anti-GluA3 hIgGs. Of note, anti-GluA3 hIgGs-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission with a positive allosteric modulator of AMPA receptors. Overall, our study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and pathology and identified a specific subgroup of FTLD patients. Our findings will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Decisions that favor one's own interest versus the interest of another individual depend on context and the relationships between individuals. The neurobiology underlying selfish choices or choices that benefit others is not understood. We developed a two-choice social decision-making task in which mice can decide whether to share a reward with their conspecifics. Preference for altruistic choices was modulated by familiarity, sex, social contact, hunger, hierarchical status and emotional state matching. Fiber photometry recordings and chemogenetic manipulations demonstrated that basolateral amygdala (BLA) neurons are involved in the establishment of prosocial decisions. In particular, BLA neurons projecting to the prelimbic (PL) region of the prefrontal cortex mediated the development of a preference for altruistic choices, whereas PL projections to the BLA modulated self-interest motives for decision-making. This provides a neurobiological model of altruistic and selfish choices with relevance to pathologies associated with dysfunctions in social decision-making., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Despite being a crucial physiological function of the brain, the mechanisms underlying forgetting are still poorly understood. Estrogens play a critical role in different brain functions, including memory. However, the effects of sex hormones on forgetting vulnerabilitymediated by retroactive interference (RI), a phenomenon in which newly acquired information interferes with the retrieval of already stored information, are still poorly understood. The aim of our study was to characterize the sex differences in interference-mediated forgetting and identify the underlying molecular mechanisms. We found that adult male C57bl/6 mice showed a higher susceptibility to RI-dependent memory loss than females. The preference index (PI) in the NOR paradigm was 52.7 ± 5.9% in males and 62.3 ± 13.0% in females. The resistance to RI in female mice was mediated by estrogen signaling involving estrogen receptor α activation in the dorsal hippocampus. Accordingly, following RI, females showed higher phosphorylation levels (+30%) of extracellular signal-regulated kinase1/2 (ERK1/2) in the hippocampus. Pharmacological inhibition of ERK1/2 made female mice prone to RI. The PI was 70.6 ± 11.0% in vehicle-injected mice and 47.4 ± 10.8% following PD98059 administration. Collectively, our data suggest that hippocampal estrogen α receptor-ERK1/2 signaling is critically involved in a pattern separation mechanism that inhibits object-related RI in female mice.

Neural stem and progenitor cell (NSPC) depletion may play a crucial role in the cognitive impairment observed in many age-related non-communicable diseases. Insulin resistance affects brain functions through a plethora of mechanisms that remain poorly understood. In an experimental model of insulin resistant NSPCs, we identified a novel molecular circuit relying on insulin receptor substrate-1 (IRS-1)/ Forkhead box O (FoxO) signaling cascade and inhibiting the recruitment of transcription factors FoxO1 and FoxO3a on the promoters of genes regulating proliferation and self-renewal. Insulin resistance also epigenetically increased the expression of cyclin-dependent kinase inhibitor 1 (p21) and accelerated NSPC senescence. Of note, we found that stimulation of NSPCs with NSPC-derived exosomes (exo-NSPC) rescued IRS-1/FoxO activation and counteracted both the reduced proliferation and senescence of stem cells. Accordingly, intranasal administration of exo-NSPC counteracted the high-fat diet-dependent impairment of adult hippocampal neurogenesis in mice by restoring the balance between proliferating and senescent NSPCs in the hippocampus. Our findings suggest a novel mechanism underlying the metabolic control of NSPC fate potentially involved in the detrimental effects of metabolic disorders on brain plasticity. In addition, our data highlight the role of extracellular vesicle-mediated signals in the regulation of cell fate within the adult neurogenic niche., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Autoantibodies targeting the GluA3 subunit of AMPA receptors (AMPARs) have been found in patients with Rasmussen's encephalitis and different types of epilepsy and were associated with the presence of learning and attention deficits. Our group recently identified the presence of anti-GluA3 immunoglobulin G (IgG) in about 25% of patients with frontotemporal dementia (FTD), thus suggesting a novel pathogenetic role also in chronic neurodegenerative diseases. However, the in vivo behavioral, molecular and morphological effects induced these antibodies are still unexplored. We injected anti-GluA3 IgG purified from the serum of FTD patients, or control IgG, in mice by intracerebroventricular infusion. Biochemical analyses showed a reduction of synaptic levels of GluA3-containing AMPARs in the prefrontal cortex (PFC), and not in the hippocampus. Accordingly, animals injected with anti-GluA3 IgG showed significant changes in recognition memory and impairments in social behavior and in social cognitive functions. As visualized by confocal imaging, functional outcomes were paralleled by profound alterations of dendritic spine morphology in the PFC. All observed behavioral, molecular and morphological alterations were transient and not detected 10-14 days from anti-GluA3 IgG injection. Overall, our in vivo preclinical data provide novel insights into autoimmune encephalitis associated with anti-GluA3 IgG and indicate an additional pathological mechanism affecting the excitatory synapses in FTD patients carrying anti-GluA3 IgG that could contribute to clinical symptoms., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Matriptase and hepsin belong to the family of type II transmembrane serine proteases (TTSPs). Increased activity of these and the plasma protease, hepatocyte growth factor activator (HGFA), is associated with unregulated cell signaling and tumor progression through increased MET and RON kinase signaling pathways. These proteases are highly expressed in multiple solid tumors and hematological malignancies. Herein, we detail the synthesis and structure-activity relationships (SAR) of a dipeptide library bearing Arg α-ketobenozothiazole (kbt) warheads as novel inhibitors of HGFA, matriptase, and hepsin. We elucidated the substrate specificity for HGFA using positional scanning of substrate combinatorial libraries (PS-SCL), which was used to discover selective inhibitors of matriptase and hepsin. Using these selective inhibitors, we have clarified the specific role of hepsin in maintaining epithelial cell membrane integrity, known to be lost in breast cancer progression. These selective compounds are useful as chemical biology tools and for future drug discovery efforts.

Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naïve human antibody repertoire to discover a F ab (3A6) that recognizes the terminal thioesterase (TE) domain of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE-3A6 complex was determined at 2.45 Å resolution, resulting in atomic characterization of this protein-protein recognition mechanism. F ab binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Altogether, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function.

Studies on IgA nephropathy (IgAN) have identified, through GWAS, linkage analysis, and pathway scanning, molecular defects in familial and sporadic IgAN patients. In our previous study, we identified a novel variant in the SPRY2 gene that segregates with the disease in one large family. The functional characterization of this variant led us to discover that the MAPK/ERK pathway was defective not only in this family, but also in two sporadic IgAN patients wild type for SPRY2. In the present study, we have deepened the molecular analysis of the MAPK/ERK pathway and extended our evaluation to a larger cohort of sporadic patients and to one additional family. We found that the ERK pathway is defective in IgAN patients and in patients affected by another IgA-mediated disorder, Henoch-Schönlein purpura (HSP). Furthermore, we found that two other proteins, PARP1 and DNMT1, respectively involved in DNA repair and in antibody class switching and methylation maintenance duties, were critically downregulated in IgAN and HSP patients. This study opens up the possibility that defective ERK activation, in some patients, leads to PARP1 and DNMT1 downregulation suggesting that IgAN could be the consequence of a dysregulated epigenetic maintenance leading to the upregulation of several genes. In particular, PARP1 could be used as a potential biomarker for the disease.

Catalytic modules of assembly-line polyketide synthases (PKSs) have previously been observed in two very different conformations-an "extended" architecture and an "arch-shaped" architecture-although the catalytic relevance of neither has been directly established. By the use of a fully human naïve antigen-binding fragment (F ab ) library, a high-affinity antibody was identified that bound to the extended conformation of a PKS module, as verified by X-ray crystallography and tandem size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS). Kinetic analysis proved that this antibody-stabilized module conformation was fully competent for catalysis of intermodular polyketide chain translocation as well as intramodular polyketide chain elongation and functional group modification of a growing polyketide chain. Thus, the extended conformation of a PKS module is fully competent for all of its essential catalytic functions.

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

The immunoproteasome (iP) has been proposed to perform specialized roles in MHC class I antigen presentation, cytokine modulation, and T cell differentiation and has emerged as a promising therapeutic target for autoimmune disorders and cancer. However, divergence in function between the iP and the constitutive proteasome (cP) has been unclear. A global peptide library-based screening strategy revealed that the proteasomes have overlapping but distinct substrate specificities. Differing iP specificity alters the quantity of production of certain MHC I epitopes but does not appear to be preferentially suited for antigen presentation. Furthermore, iP specificity was found to have likely arisen through genetic drift from the ancestral cP. Specificity differences were exploited to develop isoform-selective substrates. Cellular profiling using these substrates revealed that divergence in regulation of the iP balances its relative contribution to proteasome capacity in immune cells, resulting in selective recovery from inhibition. These findings have implications for iP-targeted therapeutic development.

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors., Competing Interests: The authors have declared that no competing interests exist.

Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

To date, human African trypanosomiasis (HAT) still threatens millions of people throughout sub-Sahara Africa, and new approaches to disease prevention and treatment remain a priority. It is commonly accepted that HAT is fatal unless treatment is provided. However, despite the well-described general symptoms of disease progression during distinct stages of the infection, leading to encephalitic complications, coma and death, a substantial body of evidence has been reported suggesting that natural acquired immunity could occur. Hence, if under favorable conditions natural infections can lead to correct immune activation and immune protection against HAT, the development of an effective anti-HAT vaccine should remain a central goal in the fight against this disease. In this review, we will (1) discuss the vaccine candidates that have been proposed over the past years, (2) highlight the main obstacles that an efficient anti-trypanosomiasis vaccine needs to overcome and (3) critically reflect on the validity of the widely used murine model for HAT.