Investigating the role of glial cells in neurodegenerative disorders by depleting astrocytes and oligodendrocytes in a model of amyloid-beta induced cytotoxicity in organotypic hippocampal slice cultures

The etiology of Alzheimer’s disease, the most common cause of dementia, is still under debate. On the molecular level, AD is characterized by the accumulation of the beta-Amyloid protein as well as neurofibrillary tangles. Additionally, evidence pinpoints to a crucial role of inflammatory processes in the progression of the disease. The cellular mechanisms leading to neuroinflammation and neuronal death observed in Alzheimer’s disease are still unclear. It has long been thought that the immune system of the brain is unique and separated from the blood and the remaining organism by the blood brain barrier. Actually, the central nervous system has a special set of defence mechanisms against pathogens and invaders that is distinct from the innate or adaptive immune system. Important players of this defence system are glial cells. Glial cells are non-neuronal cells that are subdivided into three cell types, including microglia, astrocytes and oligodendrocytes, with different modes of action and function. Astrocytes and microglia actively take part in the defence process, whereas oligodendrocytes seem to have a more supportive function. To understand and characterize the role of glial cells in neuroinflammatory processes, it is crucial to establish a model system that displays the pathology of Alzheimer’s disease and allows the individual study of cells. It was already shown that microglia could successfully be depleted using the bisphosphate clodronate from organotypic hippocampal slice cultures. However, there is still a lack of a system to study the individual role of astrocytes and oligodendrocytes ex vivo. Here, two different approaches were tested on their ability to be useful in ablation studies of astrocytes and oligodendrocytes in vitro and ex vivo. For in vitro studies primary derived cells from either embryonic or postnatal mice were used. Ex vivo studies were exerted on murine organotypic hippocampal slice cultures that in general serve as an excellent model system to study cell to cell interactions and communications. The depletion of astrocytes by antibody-toxin conjugates was promising in vitro but lacking efficacy ex vivo. The cell surface transporter GLAST-1 was found to be a promising candidate, since internalization signals were strongest and treatment of slices responded in a concentration-dependent manner. Treatment of slices with the L-alpha-aminoadipic acid led to depletion of astrocytes and provides evidence for their impor-tance in maintaining cytoarchitecture and thereby influenc-ing the viability of neuronal cells. Antibody-toxin conjugates were also used for the ablation of Oli-neu cells in vitro. Among them, a combination containing the antibody against the cell surface protein Claudin-11 showed greater effects on the viability than the combination of the receptor antibody GPR17. The underlying processes for the internalization are of great interest since this method could be further developed for drug-delivery studies to different specific cells. This method could lead to a broader spectrum of ways for drug-targeting as it is used in cancer treatment

The neuroprotective role of microglial cells against amyloid beta-mediated toxicity in organotypic hippocampal slice cultures

During Alzheimer’s disease (AD) progression, microglial cells play complex roles and have potentially detrimental as well as beneficial effects. The use of appropriate model systems is essential for characterizing and understanding the roles of microglia in AD pathology. Here, we used organotypic hippocampal slice cultures (OHSCs) to investigate the impact of microglia on amyloid beta (Aβ)-mediated toxicity. Neurons in OHSCs containing microglia were not vulnerable to cell death after 7 days of repeated treatment with Aβ1-42 oligomer-enriched preparations. However, when clodronate was used to remove microglia, treatment with Aβ1-42 resulted in significant neuronal death. Further investigations indicated signs of endoplasmic reticulum stress and caspase activation after Aβ1-42 challenge only when microglia were absent. Interestingly, microglia provided protection without displaying any classic signs of activation, such as an amoeboid morphology or the release of pro-inflammatory mediators (e.g., IL-6, TNF-α, NO). Furthermore, depleting microglia or inhibiting microglial uptake mechanisms resulted in significant more Aβ deposition compared to that observed in OHSCs containing functional microglia, suggesting that microglia efficiently cleared Aβ. Because inhibiting microglial uptake increased neuronal cell death, the ability of microglia to engulf Aβ is thought to contribute to its protective properties. Our study argues for a beneficial role of functional ramified microglia whereby they act against the accumulation of neurotoxic forms of Aβ and support neuronal resilience in an in situ model of AD pathology

Microglial Transforming Growth Factor-β Signaling in Alzheimer’s Disease

Vidovic N, Spittau B. Microglial Transforming Growth Factor-β Signaling in Alzheimer’s Disease. International Journal of Molecular Sciences. 2024;25(6): 3090.Novel technologies such as single-cell RNA and single-nucleus RNA sequencing have shed new light on the complexity of different microglia populations in physiological and pathological states. The transcriptomic profiling of these populations has led to the subclassification of specific disease-associated microglia and microglia clusters in neurodegenerative diseases. A common profile includes the downregulation of homeostasis and the upregulation of inflammatory markers. Furthermore, there is concordance in few clusters between murine and human samples. Apolipoprotein E, which has long been considered a high-risk factor for late-onset Alzheimer’s disease, is strongly regulated in both these murine and human clusters. Transforming growth factor-β plays an essential role during the development and maturation of microglia. In a pathological state, it attenuates their activation and is involved in numerous cell regulatory processes. Transforming growth factor-β also has an influence on the deposition of amyloid-beta, as it is involved in the regulation of key proteins and molecules. Taken together, this review highlights the complex interaction of apolipoprotein E, the triggering receptor on myeloid cells 2, and transforming growth factor-β as part of a regulatory axis in microglia at the onset and over the course of Alzheimer’s disease

The Influence of TGFß signaling on the phagocytosis of amyloid beta species

Vidovic N, Kryzan O, Kühle M, Mehlhaff M, Spittau B. The Influence of TGFß signaling on the phagocytosis of amyloid beta species. Glia. 2023;71(Suppl. 1):E313

A Custom Panel for Profiling Microglia Gene Expression

Potru P, Vidovic N, Wiemann S, Ruß T, Trautmann M, Spittau B. A Custom Panel for Profiling Microglia Gene Expression. Cells. 2024;13(7): 630.Despite being immune cells of the central nervous system (CNS), microglia contribute to CNS development, maturation, and homeostasis, and microglia dysfunction has been implicated in several neurological disorders. Recent advancements in single-cell studies have uncovered unique microglia-specific gene expression. However, there is a need for a simple yet elegant multiplexed approach to quantifying microglia gene expression. To address this, we have designed a NanoString nCounter technology-based murine microglia-specific custom codeset comprising 178 genes. We analyzed RNA extracted from ex vivo adult mouse microglia, primary mouse microglia, the BV2 microglia cell line, and mouse bone marrow monocytes using our custom panel. Our findings reveal a pattern where homeostatic genes exhibit heightened expression in adult microglia, followed by primary cells, and are absent in BV2 cells, while reactive markers are elevated in primary microglia and BV2 cells. Analysis of publicly available data sets for the genes present in the panel revealed that the panel could reliably reflect the changes in microglia gene expression in response to various factors. These findings highlight that the microglia panel used offers a swift and cost-effective means to assess microglial cells and can be used to study them in varying contexts, ranging from normal homeostasis to disease models

Effects of a Multimerized Recombinant Autoantibody Against Amyloid-β

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

The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis.

BACKGROUND: An isoleucine>methionine mutation at position 148 in the PNPLA3 gene (p.I148M, rs738409) has recently been identified as a susceptibility factor for liver damage in steatohepatitis. Here, we studied whether the PNPLA3 rs738409 polymorphism also affects predisposition to hepatocellular carcinoma (HCC). METHODS: We compared distributions of PNPLA3 genotypes in 80 and 81 Caucasian patients with alcoholic and hepatitis C virus (HCV)-associated HCC to 80 and 81 age- and sex-matched patients with alcohol-related and HCV-related cirrhosis without HCC, respectively. PNPLA3 genotypes in 190 healthy individuals from the same population served as reference. Potential confounders obesity, diabetes, HCV genotype and HBV co-infection were controlled by univariate and multivariate logistic regression with forward variable selection. RESULTS: PNPLA3 genotypes were in Hardy-Weinberg equilibrium for all study groups. The frequency of the 148M allele was significantly (p<0.001) increased in alcoholic cirrhosis with (53.7%) and without HCC (36.2%) but was not different between healthy controls (22.9%) and patients with cirrhosis (25.3%; p = 0.545) and HCC (30.2%; p = 0.071) due to hepatitis C. HCC risk was highest in 148M/M homozygous patients with alcoholic liver disease (odds ratio (OR) 16.8 versus healthy controls; 95% confidence interval (CI) 6.68-42.43, p<0.001). Finally, multivariate regression confirmed 148M/M homozygosity (OR 2.8; 95%-CI: 1.24-6.42; p = 0.013) as HCC risk factor in alcoholic cirrhosis. In HCV-related cirrhosis only HCV genotype 1 was confirmed as a HCC risk factor (OR 4.2; 95%-CI: 1.50-11.52; p = 0.006). CONCLUSION: The PNPLA3 148M variant is a prominent risk factor for HCC in patients with alcoholic cirrhosis, while its effects are negligible in patients with cirrhosis due to HCV. This polymorphism provides an useful tool to identify individuals with particularly high HCC risk in patients with alcoholic liver disease that should be taken into account in future HCC prevention studies

Demographic and Clinical Data of the Study Groups.

<p>Demographic and Clinical Data of the Study Groups.</p