Absence of microglia promotes diverse pathologies and early lethality in Alzheimer's disease mice.

Microglia are strongly implicated in the development and progression of Alzheimer's disease (AD), yet their impact on pathology and lifespan remains unclear. Here we utilize a CSF1R hypomorphic mouse to generate a model of AD that genetically lacks microglia. The resulting microglial-deficient mice exhibit a profound shift from parenchymal amyloid plaques to cerebral amyloid angiopathy (CAA), which is accompanied by numerous transcriptional changes, greatly increased brain calcification and hemorrhages, and premature lethality. Remarkably, a single injection of wild-type microglia into adult mice repopulates the microglial niche and prevents each of these pathological changes. Taken together, these results indicate the protective functions of microglia in reducing CAA, blood-brain barrier dysfunction, and brain calcification. To further understand the clinical implications of these findings, human AD tissue and iPSC-microglia were examined, providing evidence that microglia phagocytose calcium crystals, and this process is impaired by loss of the AD risk gene, TREM2.

The P522R protective variant of PLCG2 promotes the expression of antigen presentation genes by human microglia in an Alzheimer's disease mouse model.

The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology remains unknown. Chimeric AD and wild-type mice were generated by transplanting PLCG2-P522R or isogenic wild-type human induced pluripotent stem cell microglia. At 7 months of age, single-cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2-P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune-intact AD mice further demonstrated that the PLCG2-P522R variant promotes the recruitment of CD8+ T cells to the brain. These data provide the first evidence that the PLCG2-P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains.

The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C.

Lysosome dysfunction is a shared feature of rare lysosomal storage diseases and common age-related neurodegenerative diseases. Microglia, the brain-resident macrophages, are particularly vulnerable to lysosome dysfunction because of the phagocytic stress of clearing dying neurons, myelin, and debris. CD22 is a negative regulator of microglial homeostasis in the aging mouse brain, and soluble CD22 (sCD22) is increased in the cerebrospinal fluid of patients with Niemann-Pick type C disease (NPC). However, the role of CD22 in the human brain remains unknown. In contrast to previous findings in mice, here, we show that CD22 is expressed by oligodendrocytes in the human brain and binds to sialic acid­dependent ligands on microglia. Using unbiased genetic and proteomic screens, we identify insulin-like growth factor 2 receptor (IGF2R) as the binding partner of sCD22 on human myeloid cells. Targeted truncation of IGF2R revealed that sCD22 docks near critical mannose 6-phosphate­binding domains, where it disrupts lysosomal protein trafficking. Interfering with the sCD22-IGF2R interaction using CD22 blocking antibodies ameliorated lysosome dysfunction in human NPC1 mutant induced pluripotent stem cell­derived microglia-like cells without harming oligodendrocytes in vitro. These findings reinforce the differences between mouse and human microglia and provide a candidate microglia-directed immunotherapeutic to treat NPC.

Plaque-associated human microglia accumulate lipid droplets in a chimeric model of Alzheimer's disease.

BACKGROUND: Disease-associated microglia (DAMs), that surround beta-amyloid plaques, represent a transcriptionally-distinct microglial profile in Alzheimer's disease (AD). Activation of DAMs is dependent on triggering receptor expressed on myeloid cells 2 (TREM2) in mouse models and the AD TREM2-R47H risk variant reduces microglial activation and plaque association in human carriers. Interestingly, TREM2 has also been identified as a microglial lipid-sensor, and recent data indicates lipid droplet accumulation in aged microglia, that is in turn associated with a dysfunctional proinflammatory phenotype. However, whether lipid droplets (LDs) are present in human microglia in AD and how the R47H mutation affects this remains unknown. METHODS: To determine the impact of the TREM2 R47H mutation on human microglial function in vivo, we transplanted wild-type and isogenic TREM2-R47H iPSC-derived microglial progenitors into our recently developed chimeric Alzheimer mouse model. At 7 months of age scRNA-seq and histological analyses were performed. RESULTS: Here we report that the transcriptome of human wild-type TREM2 and isogenic TREM2-R47H DAM xenografted microglia (xMGs), isolated from chimeric AD mice, closely resembles that of human atherosclerotic foam cells. In addition, much like foam cells, plaque-bound xMGs are highly enriched in lipid droplets. Somewhat surprisingly and in contrast to a recent in vitro study, TREM2-R47H mutant xMGs exhibit an overall reduction in the accumulation of lipid droplets in vivo. Notably, TREM2-R47H xMGs also show overall reduced reactivity to plaques, including diminished plaque-proximity, reduced CD9 expression, and lower secretion of plaque-associated APOE. CONCLUSIONS: Altogether, these results indicate lipid droplet accumulation occurs in human DAM xMGs in AD, but is reduced in TREM2-R47H DAM xMGs, as it occurs secondary to TREM2-mediated changes in plaque proximity and reactivity.

PapRIV, a BV-2 microglial cell activating quorum sensing peptide.

Quorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood-brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

Stem-cell-derived human microglia transplanted in mouse brain to study human disease.

Although genetics highlights the role of microglia in Alzheimer's disease, one-third of putative Alzheimer's disease risk genes lack adequate mouse orthologs. Here we successfully engraft human microglia derived from embryonic stem cells in the mouse brain. The cells recapitulate transcriptionally human primary microglia ex vivo and show expression of human-specific Alzheimer's disease risk genes. Oligomeric amyloid-ß induces a divergent response in human versus mouse microglia. This model can be used to study the role of microglia in neurological diseases.

Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo.

iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Aß-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Aß-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in vivo function of patient-derived and genetically modified microglia.

Human stem cell-derived monocytes and microglia-like cells reveal impaired amyloid plaque clearance upon heterozygous or homozygous loss of TREM2.

INTRODUCTION: Murine microglia expressing the Alzheimer's disease-linked TREM2R47H mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2+/R47H human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia-like cells (tMGs) to examine the effect of the TREM2+/R47H mutation and loss of TREM2 on phagocytosis. METHODS: We generated isogenic TREM2+/R47H, TREM2+/-, and TREM2-/- hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1+/- mouse brains. RESULTS: We demonstrated that tMGs resemble cultured human microglia. TREM2+/- and TREM2-/- hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild-type hPSC progeny, with no difference for TREM2+/R47H progeny. DISCUSSION: In vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2+/R47H mutation but was significantly impaired in TREM2+/- and TREM2-/- progeny.

Generating tissue-resident macrophages from pluripotent stem cells: Lessons learned from microglia.

Over the past decades, the importance of the immune system in a broad scope of pathologies, has drawn attention towards tissue-resident macrophages, such as microglia in the brain. To enable the study of for instance microglia, it is crucial to recreate in vitro (and in vivo) assays. However, very fast loss of tissue-specific features of primary tissue resident macrophages, including microglia, upon in vitro culture has complicated such studies. Moreover, limited knowledge of macrophage developmental pathways and the role of local 'niche factors', has hampered the generation of tissue-resident macrophages from pluripotent stem cells (PSC). Recent data on the ontogeny of tissue-resident macrophages, combined with bulk and single cell RNAseq studies have identified the distinct origins and gene profile of microglia compared to other myeloid cells. As a result, over the past years, protocols have been published to create hPSC-derived microglia-'like' cells, as these cells are considered potential new therapeutic targets for therapies to treat neurodegenerative diseases. In this review we will provide an overview of different approaches taken to generate human microglia in vitro, taking into account their origin, and resemblance to their in vivo counterpart. Finally, we will discuss cell-extrinsic (culture conditions) and intrinsic factors (transcriptional machinery and epigenetics) that we believe can improve future differentiation protocols of tissue-resident macrophages from stem cells.

Stem cell-derived hepatocytes: A novel model for hepatitis E virus replication.

BACKGROUND & AIMS: Yearly, approximately 20million people become infected with the hepatitis E virus (HEV) resulting in over 3million cases of acute hepatitis. Although HEV-mediated hepatitis is usually self-limiting, severe cases of fulminant hepatitis as well as chronic infections have been reported, resulting annually in an estimated 60,000 deaths. We studied whether pluripotent stem cell (PSC)-derived hepatocytes, mesodermal and/or neuroprogenitor cells support HEV replication. METHODS: Human PSC were differentiated towards hepatocyte-like cells, mesodermal cells and neuroprogenitors and subsequently infected with HEV. Infection and replication of HEV was analyzed by qRT-PCR, RNA in situ hybridization, negative strand RT-PCR, production of infectious virions and transfection with a transient HEV reporter replicon. RESULTS: PSC-derived hepatocytes supported the complete replication cycle of HEV, as demonstrated by the intracellular presence of positive and negative strand HEV RNA and the production of infectious virions. The replication of the virus in these cells was inhibited by the antiviral drugs ribavirin and interferon-α2b. In contrast to PSC-derived hepatocytes, PSC-derived mesodermal cells and neuroprogenitors only supported HEV replication upon transfection with a HEV subgenomic replicon. CONCLUSION: We demonstrate that PSC can be used to study the hepatotropism of HEV infection. The complete replication cycle of HEV can be recapitulated in infected PSC-derived hepatocytes. By contrast other germ layer cells support intracellular replication but are not infectable with HEV. Thus the early steps in the viral cycle are the main determinant governing HEV tissue tropism. PSC-hepatocytes offer a physiological relevant tool to study the biology of HEV infection and replication and may aid in the design of therapeutic strategies.