The discovery of TREM2 as a myeloid-specific Alzheimer's disease (AD) risk gene has accelerated research into the role of microglia in AD. While TREM2 mouse models have provided critical insight, the ...normal and disease-associated functions of TREM2 in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we find that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1α/CXCR4-mediated chemotaxis, culminating in an impaired response to beta-amyloid plaques in vivo. Single-cell sequencing of xenotransplanted human microglia further highlights a loss of disease-associated microglial (DAM) responses in human TREM2 knockout microglia that we validate by flow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles in the development and progression of AD.
The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are ...associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca
signaling have been observed in several AD models, we investigated TREM2 regulation of Ca
signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca
signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y
and P2Y
receptors, results in greater release of Ca
from the endoplasmic reticulum stores, which triggers sustained Ca
influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca
probe, Salsa6f, we found that cytosolic Ca
tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca
using a P2Y
receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca
response to purinergic signals, suggesting a window of Ca
signaling for optimal microglial motility.
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.
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.
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.
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.
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.
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•Absence of microglia in Alzheimer’s disease mice promotes cerebral amyloid angiopathy•Increased vascular amyloid is accompanied by hemorrhages, calcification, and lethality•Adult transplantation of microglia prevents these diverse pathological changes•snRNA-seq reveals the impact of microglial absence and replacement on other cell types
Kiani Shabestari et al. show that genetic absence of microglia in Alzheimer’s disease mice causes a shift from parenchymal amyloid plaques to cerebral amyloid angiopathy, brain calcification and hemorrhages, and premature lethality. Adult microglial transplantation rescues these pathological changes, demonstrating that microglia protect the brain against detrimental Alzheimer’s disease co-pathologies.
Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning ...neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs.
Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR.
Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers.
Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders.
Basic science.
Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE
Aβ) is a highly pathogenic molecule with increased neurotoxicity and ...propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE
Aβ represents a major constituent of the amyloid plaque. The data show that pE
Aβ formation is increased at early pre-symptomatic disease stages, while tau phosphorylation and aggregation mostly occur at later stages of the disease. This suggests that pE
Aβ accumulation may be an early event in the disease pathogenesis and can be prophylactically targeted to prevent the onset of AD. The vaccine (AV-1986R/A) was generated by chemically conjugating the pE
Aβ
fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in Advax
adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 10
-10
against pE
Aβ and 10
-10
against the full-sized peptide in the 5XFAD AD mouse model. The vaccination showed efficient clearance of the pathology, including non-pyroglutamate-modified plaques, from the mice brains. AV-1986R/A is a novel promising candidate for the immunoprevention of AD. It is the first late preclinical candidate which selectively targets a pathology-specific form of amyloid with minimal immunoreactivity against the full-size peptide. Successful translation into clinic may offer a new avenue for the prevention of AD via vaccination of cognitively unimpaired individuals at risk of disease.
Risk on the Table Creager, Angela N. H; Gaudillière, Jean-Paul
01/2021, Letnik:
21
eBook
Over the last century, the industrialization of agriculture and
processing technologies have made food abundant and relatively
inexpensive for much of the world's population. Simultaneously,
...pesticides, nitrates, and other technological innovations intended
to improve the food supply's productivity and safety have generated
new, often poorly understood risks for consumers and the
environment. From the proliferation of synthetic additives to the
threat posed by antibiotic-resistant bacteria, the chapters in
Risk on the Table zero in on key historical cases in North
America and Europe that illuminate the history of food safety,
highlighting the powerful tensions that exists among scientific
understandings of risk, policymakers' decisions, and cultural
notions of "pure" food.
Mouse epiblast stem cells (mEpiSCs) and human embryonic stem cells (hESCs) are primed pluripotent stem cells whose self-renewal can be maintained through cytoplasmic stabilization and retention of ...β-catenin. The underlying mechanism, however, remains largely unknown. Here, we show that cytoplasmic β-catenin interacts with and retains TAZ, a Hippo pathway effector, in the cytoplasm. Cytoplasmic retention of TAZ promotes mEpiSC self-renewal in the absence of nuclear β-catenin, whereas nuclear translocation of TAZ induces mEpiSC differentiation. TAZ is dispensable for naive mouse embryonic stem cell (mESC) self-renewal but required for the proper conversion of mESCs to mEpiSCs. The self-renewal of hESCs, like that of mEpiSCs, can also be maintained through the cytoplasmic retention of β-catenin and TAZ. Our study indicates that how TAZ regulates cell fate depends on not only the cell type but also its subcellular localization.
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•TAZ is a binding partner of cytoplasmic β-catenin•TAZ is essential for the conversion of mESCs to mEpiSCs•Cytoplasmic retention of TAZ promotes mEpiSC and hESC self-renewal•Nuclear translocation of TAZ induces mEpiSC and hESC differentiation
In this article, Qi-Long Ying and colleagues show that cytoplasmic β-catenin interacts and retains TAZ in the cytoplasm in mEpiSCs and hESCs. Cytoplasmic retention of TAZ promotes mEpiSC and hESC self-renewal in the absence of nuclear β-catenin, whereas nuclear translocation of TAZ induces mEpiSC and hESC differentiation. This study demonstrates that transcriptional co-activators can also exert functional roles in the cytoplasm.
Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a ...protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia.
For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1
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•Protocol for ribonucleoprotein-mediated CRISPR/Cas9 editing of iPSCs•Generation and differentiation of CSF1R-G795A iPSC line into HPCs and iPSC-microglia•Intracranial transplantation of G795A-HPCs or G795A-iMG into xenotolerant mice•Complete CSF1Ri-mediated microglia replacement
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia.
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