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Pathologically altered tau protein is a common denominator of neurodegenerative disorders including Alzheimer’s disease (AD) and other tauopathies. Therefore, promising immunotherapeutic approaches target and eliminate extracellular pathogenic tau species, which are thought to be responsible for seeding and propagation of tau pathology. Tau isoforms in misfolded states can propagate disease pathology in a template-dependent manner, proposed to be mediated by the release and internalization of extracellular tau. Monoclonal antibody DC8E8, binding four highly homologous and independent epitopes in microtubule-binding domain (MTBD) of diseased tau, inhibits tau-tau interaction, discriminates between healthy and pathologically truncated tau and reduces tau pathology in animal model in vivo. Here, we show that DC8E8 antibody acts via extracellular mechanism and does not influence viability and physiological functions of neurons. Importantly, in vitro functional assays showed that DC8E8 recognises pathogenic tau proteins of different size and origin, and potently blocks their entry into neurons. Next, we examined the mechanisms by which mouse antibody DC8E8 and its humanized version AX004 effectively block the neuronal internalization of extracellular AD tau species. We determined a novel mode of action of a therapeutic candidate antibody, which potently inhibits neuronal internalization of AD tau species by masking of epitopes present in MTBD important for interaction with neuron surface Heparan Sulfate Proteoglycans (HSPGs). We show that interference of tau-heparane sulfate interaction with DC8E8 antibody via steric hindrance represents an efficient and important therapeutic approach halting tau propagation.

Current understanding of how immunodeficiencies impact protective responses against viral infections and vaccination is primarily derived from adult cohorts that may not accurately reflect the pediatric, adolescent, and young adult population. This cross-sectional study aimed to evaluate immune responses in this underrepresented population affected by various immunodeficiencies after SARS-CoV-2 infection, two doses of the mRNA BNT162b2 vaccine, or after a combination of both. We analyzed blood samples from 102 immunocompromised patients (IC) (5–25 years) categorized into groups of primary immunodeficiencies (PID, n=17), bronchial asthma and allergic rhinitis (BA-AR, n=39), rheumatoid diseases (RD, n=21), and individuals who had undergone hematopoietic stem cell transplantation (HSCT, n=28), as well as 30 healthy individuals (9–26 years). We measured titres of Spike-specific IgM, IgA, and IgG antibody classes (including IgG subclasses) in plasma using ELISA and evaluated their inhibitory potential in a Spike-ACE2 cell-based internalization assay. Spike-specific CD4 T-cells were examined using a flow cytometry-based proliferation assay (FASCIA). In the IC group, all participants except eight generated detectable levels of IgG antibodies. The IgG titres induced by vaccination (Geometric mean titre (GMT vac ) = 205023, 95% CI: 116074-362136) and a combination of vaccination and infection (GMT hyb = 172819, 95% CI: 33133-901403) were higher than after infection (GMT inf = 3323, 95% CI: 578-19109, P vac/inf = .006 and P hyb/inf = .001). On the other hand, the hybrid immunity induced the highest IgA titres (GMT hyb = 2672, 95% CI: 566-12623) compared to vaccination (GMP vac = 275, 95% CI: 97-777, P hyb/vac = .016) and infection (GMT inf = 60, 95% CI: 13-280, P hyb/inf = .002). The IgG titres in vaccinated and hybrid immunity groups strongly correlated (r Spearman = 0.86, P <.0001) with the levels of antibodies inhibiting the internalization of Spike protein (S protein) in a cell-based assay. Most IC patients (except five) also developed above-threshold Spike-specific CD4 T-cell responses, which were not statistically different from the responses in the healthy control group. Our data show that infection and vaccination can induce protective humoral or cellular responses against SARS-CoV-2 in IC patients. The activated cellular response in patients with agammaglobulinemia may assist them in overcoming viral infections.

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene ( Igh6) , IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

Immunotherapies targeting pathological tau have recently emerged as a promising approach for treatment of neurodegenerative disorders. We have previously showed that the mouse antibody DC8E8 discriminates between healthy and pathological tau, reduces tau pathology in murine tauopathy models and inhibits neuronal internalization of AD tau species in vitro. Here we show, that DC8E8 and antibodies elicited against the first-in-man tau vaccine, AADvac1, which is based on the DC8E8 epitope peptide, both promote uptake of pathological tau by mouse primary microglia. IgG1 and IgG4 isotypes of AX004, the humanized versions of DC8E8, accelerate tau uptake by human primary microglia isolated from post-mortem aged and diseased brains. This promoting activity requires the presence of the Fc-domain of the antibodies. The IgG1 isotype of AX004 showed greater ability to promote tau uptake compared to the IgG4 isotype, while none of the antibody-tau complexes provoked increased pro-inflammatory activity of microglia. Our data suggest that IgG1 has better suitability for therapeutic development.

Background Abnormal misfolded tau protein is a driving force of neurofibrillary degeneration in Alzheimer's disease. It has been shown that tau oligomers play a crucial role in the formation of intracellular neurofibrillary tangles. They are intermediates between soluble tau monomers and insoluble tau filaments and are suspected contributors to disease pathogenesis. Oligomeric tau can be released into the extracellular space and spread throughout the brain. This finding opens the question of whether brain macrophages or blood monocytes have the potential to phagocytose extracellular oligomeric tau. Methods We have used stable rat primary microglial cells, rat peripheral monocytes-derived macrophages, BV2 microglial and TIB67 macrophage immortalized cell lines that were challenged by tau oligomers prepared by an in vitro aggregation reaction. The efficiency of cells to phagocytose oligomeric protein was evaluated with confocal microscopy. The ability to degrade tau protein was analyzed by immunoblotting. Results Confocal microscopy analyses showed that macrophages were significantly more efficient in phagocytosing oligomerized tau proteins than microglial cells. In contrast to macrophages, microglia are able to degrade the internalized oligomeric tau only after stimulation with lipopolysaccharide (LPS). Conclusions Our data suggests that microglia may not be the principal phagocytic cells able to target extracellular oligomeric tau. We found that peripheral macrophages display a high potency for elimination of oligomeric tau and therefore could play an important role in the modulation of neurofibrillary pathology in Alzheimer's disease.

Angiotensin-converting enzyme 2 (ACE2), one of the key enzymes of the renin-angiotensin system (RAS), plays an important role in SARS-CoV-2 infection by functioning as a virus receptor. Angiotensin peptides Ang I and Ang II, the substrates of ACE2, can modulate the binding of SARS-CoV-2 Spike protein to the ACE2 receptor. In the present work, we found that co incubation of HEK-ACE2 and Vero E6 cells with the SARS-CoV-2 Spike pseudovirus (PVP) resulted in stimulation of the virus entry at low and high micromolar concentrations of Ang I and Ang II, respectively. The potency of Ang I and Ang II stimulation of virus entry corresponds to their binding affinity to ACE2 catalytic pocket with 10 times higher efficiency of Ang II. The Ang II induced mild increase of PVP infectivity at 20 µM; while at 100 µM the increase (129.74±3.99 %) was highly significant (p<0.001). Since the angiotensin peptides act in HEK ACE2 cells without the involvement of angiotensin type I receptors, we hypothesize that there is a steric interaction between the catalytic pocket of the ACE2 enzyme and the SARS-CoV-2 S1 binding domain. Oversaturation of the ACE2 with their angiotensin substrate might result in increased binding and entry of the SARS-CoV-2. In addition, the analysis of angiotensin peptides metabolism showed decreased ACE2 and increased ACE activity upon SARS-CoV-2 action. These effects should be taken into consideration in COVID-19 patients suffering from comorbidities such as the over-activated renin-angiotensin system as a mechanism potentially influencing the SARS-CoV-2 invasion into recipient cells.

Abstract Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene ( Igh6) , IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

The microtubule associated protein tau forms disease-specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to be able to control this process for drug development purposes, it is crucial to study in vitro tau aggregation methods and investigate the structures of the obtained filaments at atomic level. Here, we used the tau fragment dGAE(297-391), which aggregates spontaneously, to seed formation of full-length tau filaments. The structures of dGAE and full-length tau filaments were investigated by solid-state MAS NMR showing that dGAE allows propagating a chronic traumatic encephalopathy (CTE)-like fold to full-length tau. This work demonstrates that in vitro preparation of disease-specific types of full-length tau filaments is feasible.Competing Interest StatementThe authors have declared no competing interest.

Cognitive dysfunction syndrome (CDS) in pet animals constitutes a pressing problem of the modern society. Millions of senior dogs and cats undergo age-related behavioral changes that impact their social interactions. Currently, it is difficult to discriminate between normal aging and dementing processes. The situation is akin to that in humans; however, human medicine receives enormous resources that resulted in a set of current diagnostic criteria including a number of dementia assessing scales, diagnostic assays, and novel potential biomarkers. While animal well-being is not in the limelight of societal interest, the dementia diagnostics starts to catch up. Nevertheless, biochemical markers related to the animal dementia are underdeveloped, despite the fact that dogs and cats provide natural models for human dementia.

Recently emerged SARS-CoV-2 variants show resistance to some antibodies that were authorized for emergency use. We employed hybridoma technology combined with authentic virus assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize new variants of SARS-CoV-2. AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 and B.1.351. Finally, the combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. The neutralizing properties were fully reproduced in chimeric mouse-human versions, which may represent a promising tool for COVID-19 therapy.