Human microglia differentially respond to β-amyloid, tau, and combined Alzheimer’s disease pathologies in vivo

Recent studies have identified important species-dependent differences in the response of microglia to β-amyloid (Aβ) pathology. Yet, whether human microglia also interact differently with the pathognomonic combination of amyloid and tau pathologies that occur in Alzheimer’s disease (AD) remains unclear. We generated a xenotolerant mouse model of AD that develops both plaque and tangle pathologies, transplanted stem cell-derived microglial progenitors and examined the interactions between human microglia and AD pathologies with scRNA sequencing, immunohistochemistry, and in vitro modeling. The combined amyloid and tau pathologies induced robust type-I interferon and proinflammatory cytokine responses, as well as an increased adoption of a distinct “rod” morphology in human microglia. The rod morphology could be induced with type-I interferon treatment in vitro. We provide new insights into human microglial responses to combined AD pathologies and a novel platform to investigate and manipulate human microglia in vivo.

Highlights

• Amyloid pathology promotes the rapid development of neurofibrillary tangles and neuronal loss in a novel chimeric model of AD.
• Combined Alzheimer’s disease pathologies lead to an expansion of disease-associated microglia (DAM) and exacerbate Interferon-responsive and cytokine/chemokine-enriched states in xenotransplanted human microglia.
• The combination of amyloid and tau promotes the development of a distinctive rod microglial phenotype that closely correlates with tau pathology and neurodegeneration.
• Rod morphology and transcriptional changes can be modeled in vitro by treatment of induced pluripotent stem cells (iPSC) -microglia with type-I interferons.

CSF1R inhibitor-resistant model for CNS-wide microglia replacement strategies

Recent advances in cell-based therapies have demonstrated therapeutic safety and efficacy for a variety of diseases. However, significant challenges remain in their development for the treatment of neurological disorders. Preclinical studies have explored microglial replacement strategies utilizing bone marrow transplantation and CSF1R inhibitor (CSF1Ri) treatment. However, these approaches often require highly invasive strategies and result in engrafted cells that remain transcriptionally and functionally distinct from microglia. To assess less-invasive microglia replacement strategies capable of preserving microglial ontogeny, we developed a Csf1r-G793A knockin mouse. We found that G793A mice develop typical microglial densities and peripheral hematopoietic populations, which exhibit broad CSF1Ri resistance enabling widespread microglia replacement following direct intraparenchymal injection or bone marrow transplantation. Furthermore, we demonstrate that widespread microglia replacement can be achieved via less-invasive intracisternal delivery of CSF1Ri-resistant murine and induced pluripotent stem cell (iPSC)-derived human microglia. Together with the accompanying study by Lombroso and colleagues, our findings demonstrate that G793A mice provide a robust and broadly applicable source of engraftable donor microglia and peripheral macrophages for the preclinical investigation of microglia replacement strategies.

Harnessing human iPSC-microglia for CNS-wide delivery of disease-modifying proteins

Widespread delivery of therapeutic proteins to the brain remains challenging. To determine whether human induced pluripotent stem cell (iPSC)-microglia (iMG) could enable brain-wide and pathology-responsive delivery of therapeutic cargo, we utilized CRISPR gene editing to engineer iMG to express the Aβ-degrading enzyme neprilysin under control of the plaque-responsive promoter, CD9. To further determine whether increased engraftment enhances efficacy, we utilized a CSF1R-inhibitor resistance approach. Interestingly, both localized and brain-wide engraftment in Alzheimer’s disease (AD) mice reduced multiple biochemical measures of pathology. However, within the plaque-dense subiculum, reductions in plaque load, dystrophic neurites, and astrogliosis and preservation of neuronal density were only achieved following widespread microglial engraftment. Lastly, we examined chimeric models of breast cancer brain metastases and demyelination, demonstrating that iMG adopt diverse transcriptional responses to differing neuropathologies, which could be harnessed to enable widespread and pathology-responsive delivery of therapeutics to the CNS.

Highlights

Therapeutic potential of human microglia transplantation in a chimeric model of CSF1R-related leukoencephalopathy

Microglia replacement strategies are increasingly being considered for the treatment of primary microgliopathies like adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). However, available mouse models fail to recapitulate the diverse neuropathologies and reduced microglia numbers observed in patients. In this study, we generated a xenotolerant mouse model lacking the fms-intronic regulatory element (FIRE) enhancer within Csf1r, which develops nearly all the hallmark pathologies associated with ALSP. Remarkably, transplantation of human induced pluripotent stem cell (iPSC)-derived microglial (iMG) progenitors restores a homeostatic microglial signature and prevents the development of axonal spheroids, white matter abnormalities, reactive astrocytosis, and brain calcifications. Furthermore, transplantation of CRISPR-corrected ALSP-patient-derived iMG reverses pre-existing spheroids, astrogliosis, and calcification pathologies. Together with the accompanying study by Munro and colleagues, our results demonstrate the utility of FIRE mice to model ALSP and provide compelling evidence that iMG transplantation could offer a promising new therapeutic strategy for ALSP and perhaps other microglia-associated neurological disorders.

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.

TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

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 Ca²⁺ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca²⁺ 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 Ca²⁺ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y₁₂ and P2Y₁₃ receptors, results in greater release of Ca²⁺ from the endoplasmic reticulum (ER) stores, which triggers sustained Ca²⁺ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca²⁺ probe, Salsa6f, we found that cytosolic Ca²⁺ 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 Ca²⁺ using a P2Y₁₂ receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca²⁺ response to purinergic signals, suggesting a window of Ca²⁺ signaling for optimal microglial motility.

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 a 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.

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.