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Alzheimer's disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved. As promising approach passive immunization strategies have thereby come forth. In this study, we focused on naturally occurring autoantibodies against the AD-associated peptide amyloid-β. These antibodies have already reported to show beneficial functions in vitro and in mouse models of AD. However, their availability is limited due to their low abundance in peripheral blood. In a recent study, we were able to generate four recombinant antibodies against amyloid-β. In the present study, we tested these antibodies in ELISA and SPR assays for their binding behavior and by aggregation- and phagocytosis assays as functional evidences to characterize their amyloid-β-related neutralizing and clearance abilities. Further ex vivo assay on organotypic hippocampal slice cultures gave first evidence of microglial activation and inflammatory features. The tested recombinant antibodies in IgG format showed, in comparison to naturally occurring autoantibodies against amyloid-β, insufficient binding capacities and -affinities. However, after conversion of one antibody into a single chain format multimerization of the scFv-Fc construct, the investigated binding capacity and -affinity showed improvements. Further functional assays predict a protective effect of this antibody. Although, all four recombinant antibodies showed binding to amyloid-β, promising features were only detectable after conversion into a multimeric format. The multimeric scFv-Fc antibody exhibited thereby strong impact on amyloid-β clearance and inhibition of oligomerization.

Phospholipase Cβ (PLCβ)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P2-dependent processes led to strong need for biosensors of physiological PI(4,5)P2 dynamics applicable in live-cell experiments. Membrane PI(4,5)P2 can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P2 levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubbyCT) are two such sensors widely used to study PI(4,5)P2 signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP3) produced from PI(4,5)P2 through PLCβ, and tubbyCT responses do not faithfully report on PLCβ-dependent PI(4,5)P2 dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P2 dynamics in living cells. We utilized recombinant tools to manipulate PI or Gq protein-coupled receptors (GqPCR) to stimulate PLCβ signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P2 without interacting with IP3, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P2 levels, we found that ENTH-GFP had low PI(4,5)P2 affinity. Accordingly, ENTH-GFP was highly sensitive to PLCβ-dependent PI(4,5)P2 depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate GqPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P2 levels and provides an important complementation of experimentally useful reporters of PI(4,5)P2 dynamics in physiological pathways.

Immunoglobulin G (IgG) is the most abundant antibody subclass of the human circulatory system and has important functions in the adaptive immune response. On the one hand, recognition and neutralization of antigens is mediated by the fab fragment, and on the other hand, processes such as phagocytosis, complement activation and inflammatory reactions are triggered by the Fc fragment. Here, the composition of conserved N-glycans attached to asparagine 297 of the IgG CH2 domain is a major critical factor that particularly modulates the effector functions of IgG. Additional attachments of fucoses, galactoses, N-acetylglucosamines, and sialic acids have been identified as factors that influence the affinity to a wide range of complement proteins and receptors and, thus, secondarily induce the secretion of pro- and anti-inflammatory cytokines. Consequently, alterations in the IgG Fc N-glycosylation pattern can provoke disruptions in the immunological state and are accompanied by various diseases, although the involvement of changed IgG glycosylation in disease outbreaks remains unknown. In addition to many autoimmune diseases, which have already been extensively reviewed, there are a number of further disorders related to altered IgG glycosylation patterns. In the present review, we focus on neurologic diseases, as in the last few years, an increasing number of studies have been published in this field. Due to the absence of reliable early biomarkers as well as therapeutic options in many cases, such analyses are of great interest and reveal possible future approaches.

Autoreactive antibodies against the proteins alpha-synuclein (α-syn) and tau are detectable in body fluids of both healthy and diseased elderly people. However, nothing is known about their presence or titers in children. To close this gap and to characterize their temporary expression levels, we used ELISA techniques to investigate the serum titers of α-syn and tau reactive autoantibodies in 37 and 32 adults and 37 and 31 children, respectively. Most serum samples from the children exhibited both antibody types and interestingly, the levels were similar to those observed in the adult serum samples. Furthermore, sex-specific analysis revealed significantly increased α-syn reactive autoantibody titers in female children. The presence of α-syn and tau reactive autoantibodies in early childhood indicates that both immunoglobulins belong to the pool of naturally occurring autoantibodies (nAbs), as their antigen-independent synthesis from birth is a crucial characteristic. Due to their general participation in the maintenance of the physiological homeostasis, we hypothesize that both investigated nAbs are involved in the metabolic regulation of their specific antigen. Therefore, they may be a part of a mechanism that already exists in the innate immunological repertoire to provide protection from pathologies caused by dysregulated α-syn and tau metabolisms.