A Custom Panel for Profiling Microglia Gene Expression.

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.

2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro.

Background: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss.

In Operando Investigation of the Concentration Dependent NO2 Sensing Mechanism of Bi2S3 Nanorods at Low Temperatures and the Interference of O3.

The demand for gas sensors that can detect gases selectively at low temperatures has increased steadily over recent years. Most devices use semiconducting metal oxides as sensing materials which often require high operation temperatures and suffer from a lack of selectivity. Semiconducting metal sulfides were found to be a reasonable alternative for the application in sensing devices at low temperatures. Since metal sulfides are a relatively new class of materials applied in gas sensors, there is little work on sensing mechanisms and overall sensing characteristics of these materials. In this work, the authors investigated the sensing performance of Bi2S3 nanorods operated at 50 °C in the presence of several target gases and found a selective response to oxidizing gases. With the help of DC resistance measurements, diffuse reflectance infrared Fourier transform spectroscopy and work function measurements in a Kelvin Probe setup, the NO2 and O3 sensing mechanisms of Bi2S3 nanorods were revealed. While initially sulfur vacancies were the predominant reaction sites, the formation of nitrates became the key reaction in higher NO2 concentrations. Additionally, it was found that the reaction with O3 healed sulfur vacancies effectively inhibiting the reaction with NO2.

Microglial CD74 Expression Is Regulated by TGFß Signaling.

Microglia play important roles during physiological and pathological situations in the CNS. Several reports have described the expression of Cd74 in disease-associated and aged microglia. Here, we demonstrated that TGFß1 controled the expression of Cd74 in microglia in vitro and in vivo. Using BV2 cells, primary microglia cultures as well as Cx3cr1CreERT2:R26-YFP:Tgfbr2fl/fl in combination with qPCR, flow cytometry, and immunohistochemistry, we were able to provide evidence that TGFß1 inhibited LPS-induced upregulation of Cd74 in microglia. Interestingly, TGFß1 alone was able to mediate downregulation of CD74 in vitro. Moreover, silencing of TGFß signaling in vivo resulted in marked upregulation of CD74, further underlining the importance of microglial TGFß signaling during regulation of microglia activation. Taken together, our data indicated that CD74 is a marker for activated microglia and further demonstrated that microglial TGFß signaling is important for regulation of Cd74 expression during microglia activation.

Inhibition of Microglial TGFß Signaling Increases Expression of Mrc1.

Microglia are constantly surveying their microenvironment and rapidly react to impairments by changing their morphology, migrating toward stimuli and adopting gene expression profiles characterizing their activated state. The increased expression of the M2-like marker Mannose receptor 1 (Mrc1), which is also referred to as CD206, in microglia has been reported after M2-like activation in vitro and in vivo. Mrc1 is a 175-kDa transmembrane pattern recognition receptor which binds a variety of carbohydrates and is involved in the pinocytosis and the phagocytosis of immune cells, including microglia, and thought to contribute to a neuroprotective microglial phenotype. Here we analyzed the effects of TGFß signaling on Mrc1 expression in microglia in vivo and in vitro. Using C57BL/6 wild type and Cx3cr1 CreERT2 :R26-YFP:Tgfbr2 fl/fl mice-derived microglia, we show that the silencing of TGFß signaling results in the upregulation of Mrc1, whereas recombinant TGFß1 induced the delayed downregulation of Mrc1. Furthermore, chromatin immunoprecipitation experiments provided evidence that Mrc1 is not a direct Smad2/Smad4 target gene in microglia. Altogether our data indicate that the changes in Mrc1 expression after the activation or the silencing of microglial TGFß signaling are likely to be mediated by modifications of the secondary intracellular signaling events influenced by TGFß signaling.

A gold-nanoparticle stoppered [2]rotaxane.

The construction of molecular machines has captured the imagination of scientists for decades. Despite significant progress in the synthesis and studies of the properties of small-molecule components (smaller than 2-5 kilo Dalton), challenges regarding the incorporation of molecular components into real devices are still eminent. Nano-sized molecular machines operate the complex biological machinery of life, and the idea of mimicking the amazing functions using artificial nano-structures is intriguing. Both in small-molecule molecular machine components and in many naturally occurring molecular machines, mechanically interlocked molecules and structures are key functional components. In this work, we describe our initial efforts to interface mechanically-interlocked molecules and gold-nanoparticles (AuNPs); the molecular wire connecting the AuNPs is covered in an insulating rotaxane-layer, thus mimicking the macroscopic design of a copper wire. Taking advantage of recent progress in the preparation of supramolecular complexes of the cucurbit[7]uril (CB[7]) macrocycle, we have prepared a bis-thiol functionalised pseudo-rotaxane that enables us to prepare a AuNP-stoppered [2]rotaxane in water. The pseudo-rotaxane is held together extremely tightly (Ka > 1013 M-1), Ka being the association constant. We have studied the solution and gas phase guest-host chemistry using NMR spectroscopy, mass spectroscopy, and electrochemistry. The bis-thiol functionalised pseudo-rotaxane holds further a ferrocene unit in the centre of the rotaxane; this ferrocene unit enables us to address the system in detail with and without CB[7] and AuNPs using electrochemical methods.

Aged Mouse Cortical Microglia Display an Activation Profile Suggesting Immunotolerogenic Functions.

Microglia are the resident immune cells of the central nervous system (CNS) and participate in physiological and pathological processes. Their unique developmental nature suggests age-dependent structural and functional impairments that might contribute to neurodegenerative diseases. In the present study, we addressed the age-dependent changes in cortical microglia gene expression patterns and the expression of M1- and M2-like activation markers. Iba1 immunohistochemistry, isolation of cortical microglia followed by fluorescence-activated cell sorting and RNA isolation to analyze transcriptional changes in aged cortical microglia was performed. We provide evidence that aging is associated with decreased numbers of cortical microglia and the establishment of a distinct microglia activation profile including upregulation of Ifi204, Lilrb4, Arhgap, Oas1a, Cd244 and Ildr2. Moreover, flow cytometry revealed that aged cortical microglia express increased levels of Cd206 and Cd36. The data presented in the current study indicate that aged mouse cortical microglia adopt a distinct activation profile, which suggests immunosuppressive and immuno-tolerogenic functions.