EK100 and Antrodin C Improve Brain Amyloid Pathology in APP/PS1 Transgenic Mice by Promoting Microglial and Perivascular Clearance Pathways

Alzheimer’s disease (AD) is characterized by the deposition of β-amyloid peptide (Aβ). There are currently no drugs that can successfully treat this disease. This study first explored the anti-inflammatory activity of seven components isolated from Antrodia cinnamonmea in BV2 cells and selected EK100 and antrodin C for in vivo research. APPswe/PS1dE9 mice were treated with EK100 and antrodin C for one month to evaluate the effect of these reagents on AD-like pathology by nesting behavior, immunohistochemistry, and immunoblotting. Ergosterol and ibuprofen were used as control. EK100 and antrodin C improved the nesting behavior of mice, reduced the number and burden of amyloid plaques, reduced the activation of glial cells, and promoted the perivascular deposition of Aβ in the brain of mice. EK100 and antrodin C are significantly different in activating astrocytes, regulating microglia morphology, and promoting plaque-associated microglia to express oxidative enzymes. In contrast, the effects of ibuprofen and ergosterol are relatively small. In addition, EK100 significantly improved hippocampal neurogenesis in APPswe/PS1dE9 mice. Our data indicate that EK100 and antrodin C reduce the pathology of AD by reducing amyloid deposits and promoting nesting behavior in APPswe/PS1dE9 mice through microglia and perivascular clearance, indicating that EK100 and antrodin C have the potential to be used in AD treatment.

Exacerbated Age-Related Hippocampal Alterations of Microglia Morphology, ß-Amyloid and Lipofuscin Deposition and Presenilin Overexpression in Per1−/−-Mice

In humans, alterations of circadian rhythms and autophagy are linked to metabolic, cardiovascular and neurological dysfunction. Autophagy constitutes a specific form of cell recycling in many eukaryotic cells. Aging is the principal risk factor for the development of neurodegenerative diseases. Thus, we assume that both the circadian clock and autophagy are indispensable to counteract aging. We have previously shown that the hippocampus of Per1−/−-mice exhibits a reduced autophagy and higher neuronal susceptibility to ischemic insults compared to wild type (WT). Therefore, we chose to study the link between aging and loss of clock gene Per1−/−-mice. Young and aged C3H- and Per1−/−-mice were used as models to analyze the hippocampal distribution of Aβ42, lipofuscin, presenilin, microglia, synaptophysin and doublecortin. We detected several changes in the hippocampus of aged Per1−/−-mice compared to their wild type littermates. Our results show significant alterations of microglia morphology, an increase in Aβ42 deposition, overexpression of presenilin, decrease in synaptophysin levels and massive accumulation of lipofuscin in the hippocampus of 24-month-old Per1−/−-mice, without alteration of adult neurogenesis. We suggest that the marked lipofuscin accumulation, Aβ42 deposition, and overexpression of presenilin-2 observed in our experiments may be some of the consequences of the slowed autophagy in the hippocampus of aged Per1−/−-mice. This may lead during aging to excessive accumulation of misfolded proteins which may, consequently, result in higher neuronal vulnerability.

An open-source pipeline for analysing changes in microglial morphology

Changes in microglial morphology are powerful indicators of the inflammatory state of the brain. Here, we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and ‘inflammation’ training data and uses dimensionality reduction to generate a single metric of morphological change (an ‘inflammation index’). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high-fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.

Microglial Morphology Across Distantly Related Species: Phylogenetic, Environmental and Age Influences on Microglia Reactivity and Surveillance States

Microglial immunosurveillance of the brain parenchyma to detect local perturbations in homeostasis, in all species, results in the adoption of a spectrum of morphological changes that reflect functional adaptations. Here, we review the contribution of these changes in microglia morphology in distantly related species, in homeostatic and non-homeostatic conditions, with three principal goals (1): to review the phylogenetic influences on the morphological diversity of microglia during homeostasis (2); to explore the impact of homeostatic perturbations (Dengue virus challenge) in distantly related species (Mus musculus and Callithrix penicillata) as a proxy for the differential immune response in small and large brains; and (3) to examine the influences of environmental enrichment and aging on the plasticity of the microglial morphological response following an immunological challenge (neurotropic arbovirus infection). Our findings reveal that the differences in microglia morphology across distantly related species under homeostatic condition cannot be attributed to the phylogenetic origin of the species. However, large and small brains, under similar non-homeostatic conditions, display differential microglial morphological responses, and we argue that age and environment interact to affect the microglia morphology after an immunological challenge; in particular, mice living in an enriched environment exhibit a more efficient immune response to the virus resulting in earlier removal of the virus and earlier return to the homeostatic morphological phenotype of microglia than it is observed in sedentary mice.

VISTA regulates microglia homeostasis and myelin phagocytosis, and is associated with MS lesion pathology

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) that is involved in T-cell quiescence, inhibition of T-cell activation, and in myeloid cells regulates cytokine production, chemotaxis, phagocytosis, and tolerance induction. In the central nervous system (CNS), VISTA is expressed by microglia, the resident macrophage of the parenchyma, and expression is decreased during neuroinflammation; however, the function of VISTA in microglia is unknown. Here, we extensively analyzed VISTA expression in different MS lesion stages and characterized the function of VISTA in the CNS by deleting VISTA in microglia. VISTA is differentially expressed in distinct MS lesion stages. In mice, VISTA deletion in Cx3Cr1-expressing cells induced a more amoeboid microglia morphology, indicating an immune-activated phenotype. Expression of genes associated with cell cycle and immune-activation was increased in VISTA KO microglia. In response to LPS and during experimental autoimmune encephalomyelitis (EAE), VISTA KO and WT microglia shared similar transcriptional profiles and VISTA deletion did not affect EAE disease progression or microglia responses. VISTA KO in microglia in vitro decreased the uptake of myelin. This study demonstrates that VISTA is involved in microglia function, which likely affects healthy CNS homeostasis and neuroinflammation.