Particulate matter from car exhaust alters function of human iPSC-derived microglia.

BACKGROUND: Air pollution is recognized as an emerging environmental risk factor for neurological diseases. Large-scale epidemiological studies associate traffic-related particulate matter (PM) with impaired cognitive functions and increased incidence of neurodegenerative diseases such as Alzheimer's disease. Inhaled components of PM may directly invade the brain via the olfactory route, or act through peripheral system responses resulting in inflammation and oxidative stress in the brain. Microglia are the immune cells of the brain implicated in the progression of neurodegenerative diseases. However, it remains unknown how PM affects live human microglia. RESULTS: Here we show that two different PMs derived from exhausts of cars running on EN590 diesel or compressed natural gas (CNG) alter the function of human microglia-like cells in vitro. We exposed human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGLs) to traffic related PMs and explored their functional responses. Lower concentrations of PMs ranging between 10 and 100 µg ml-1 increased microglial survival whereas higher concentrations became toxic over time. Both tested pollutants impaired microglial phagocytosis and increased secretion of a few proinflammatory cytokines with distinct patterns, compared to lipopolysaccharide induced responses. iMGLs showed pollutant dependent responses to production of reactive oxygen species (ROS) with CNG inducing and EN590 reducing ROS production. CONCLUSIONS: Our study indicates that traffic-related air pollutants alter the function of human microglia and warrant further studies to determine whether these changes contribute to adverse effects in the brain and on cognition over time. This study demonstrates human iPSC-microglia as a valuable tool to study functional microglial responses to environmental agents.

Human iPSC-derived microglia carrying the LRRK2-G2019S mutation show a Parkinson's disease related transcriptional profile and function.

LRRK2-G2019S is one of the most common Parkinson's disease (PD)-associated mutations and has been shown to alter microglial functionality. However, the impact of LRRK2-G2019S on transcriptional profile of human induced pluripotent stem cell-derived microglia-like cells (iMGLs) and how it corresponds to microglia in idiopathic PD brain is not known. Here we demonstrate that LRRK2-G2019S carrying iMGL recapitulate aspects of the transcriptional signature of human idiopathic PD midbrain microglia. LRRK2-G2019S induced subtle and donor-dependent alterations in iMGL mitochondrial respiration, phagocytosis and cytokine secretion. Investigation of microglial transcriptional state in the midbrains of PD patients revealed a subset of microglia with a transcriptional overlap between the in vitro PD-iMGL and human midbrain PD microglia. We conclude that LRRK2-G2019S iMGL serve as a model to study PD-related effects in human microglia.

Single-Cell RNA-Seq Analysis of Olfactory Mucosal Cells of Alzheimer's Disease Patients.

Olfaction is orchestrated by olfactory mucosal cells located in the upper nasal cavity. Olfactory dysfunction manifests early in several neurodegenerative disorders including Alzheimer's disease, however, disease-related alterations to the olfactory mucosal cells remain poorly described. The aim of this study was to evaluate the olfactory mucosa differences between cognitively healthy individuals and Alzheimer's disease patients. We report increased amyloid-beta secretion in Alzheimer's disease olfactory mucosal cells and detail cell-type-specific gene expression patterns, unveiling 240 differentially expressed disease-associated genes compared to the cognitively healthy controls, and five distinct cell populations. Overall, alterations of RNA and protein metabolism, inflammatory processes, and signal transduction were observed in multiple cell populations, suggesting their role in Alzheimer's disease-related olfactory mucosa pathophysiology. Furthermore, the single-cell RNA-sequencing proposed alterations in gene expression of mitochondrially located genes in AD OM cells, which were verified by functional assays, demonstrating altered mitochondrial respiration and a reduction of ATP production. Our results reveal disease-related changes of olfactory mucosal cells in Alzheimer's disease and demonstrate the utility of single-cell RNA sequencing data for investigating molecular and cellular mechanisms associated with the disease.

Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids.

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

Genotyping and Frequency of PCSK9 Variations Among Hypercholesterolemic and Diabetic Subjects.

Non-synonymous single-nucleotide polymorphism (SNPs) in the gene for proprotein convertase subtilisin/kexin type 9 (PCSK9) can influence cholesterol and glucose metabolism, leading to increased risk of cardiovascular disease and diabetes. To determine the frequency of four common PCSK9 SNPs, L10Ins, A56V, I474V, and E670G, in a population sample (n = 98) of the Hail region of Kingdom of Saudi Arabia. Blood was collected from participants; serum cholesterol, blood glucose and glycated hemoglobin were determined; genomic DNA was extracted and PCR amplicons from SNP-containing PCSK9 exons were subjected to Sanger sequencing. Out of 98 participants. 10 (10.20%) carried none of the SNPs, 2 (2.04%) the L10ins/A56V linked SNPs, 35 (35.71%) the I474V SNP, 22 (22.45%) both the I474V and E670G SNPs, and 29 (29.59%) the E670G SNP. Of the 30 eucholesterolemic diabetics patients, 11 (36.66%) carried the I474V SNP, 10 (33.33%) the E679G SNP and 6 (20%) the I474V/E679G. SNPs. Of 63 diabetic patients, 26 (41.26%) carry I474V SNP and 22 (34.92%) carry E670G SNP. Our data demonstrated that the I474V and E670G PCSK9 variants are very frequent in the Hail region of Saudi Arabia and are found at even higher frequency among diabetics. Further investigations are needed to determine whether these variations or another variant segregating with them can explain its apparent association with diabetes in this population.

PSEN1ΔE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia.

Here we elucidate the effect of Alzheimer disease (AD)-predisposing genetic backgrounds, APOE4, PSEN1ΔE9, and APPswe, on functionality of human microglia-like cells (iMGLs). We present a physiologically relevant high-yield protocol for producing iMGLs from induced pluripotent stem cells. Differentiation is directed with small molecules through primitive erythromyeloid progenitors to re-create microglial ontogeny from yolk sac. The iMGLs express microglial signature genes and respond to ADP with intracellular Ca2+ release distinguishing them from macrophages. Using 16 iPSC lines from healthy donors, AD patients and isogenic controls, we reveal that the APOE4 genotype has a profound impact on several aspects of microglial functionality, whereas PSEN1ΔE9 and APPswe mutations trigger minor alterations. The APOE4 genotype impairs phagocytosis, migration, and metabolic activity of iMGLs but exacerbates their cytokine secretion. This indicates that APOE4 iMGLs are fundamentally unable to mount normal microglial functionality in AD.