Neurotoxic Microglial Activation via IFNγ-Induced Nrf2 Reduction Exacerbating Alzheimer's Disease.

Microglial neuroinflammation appears to be neuroprotective in the early pathological stage, yet neurotoxic, which often precedes neurodegeneration in Alzheimer's disease (AD). However, it remains unclear how the microglial activities transit to the neurotoxic state during AD progression, due to complex neuron-glia interactions. Here, the mechanism of detrimental microgliosis in AD by employing 3D human AD mini-brains, brain tissues of AD patients, and 5XFAD mice is explored. In the human and animal AD models, amyloid-beta (Aß)-overexpressing neurons and reactive astrocytes produce interferon-gamma (IFNγ) and excessive oxidative stress. IFNγ results in the downregulation of mitogen-activated protein kinase (MAPK) and the upregulation of Kelch-like ECH-associated Protein 1 (Keap1) in microglia, which inactivate nuclear factor erythroid-2-related factor 2 (Nrf2) and sensitize microglia to the oxidative stress and induces a proinflammatory microglia via nuclear factor kappa B (NFκB)-axis. The proinflammatory microglia in turn produce neurotoxic nitric oxide and proinflammatory mediators exacerbating synaptic impairment, phosphorylated-tau accumulation, and discernable neuronal loss. Interestingly, recovering Nrf2 in the microglia prevents the activation of proinflammatory microglia and significantly blocks the tauopathy in AD minibrains. Taken together, it is envisioned that IFNγ-driven Nrf2 downregulation in microglia as a key target to ameliorate AD pathology.

Genetic contribution to microglial activation in schizophrenia.

Several lines of evidence indicate the involvement of neuroinflammatory processes in the pathophysiology of schizophrenia (SCZ). Microglia are brain resident immune cells responding toward invading pathogens and injury-related products, and additionally, have a critical role in improving neurogenesis and synaptic functions. Aberrant activation of microglia in SCZ is one of the leading hypotheses for disease pathogenesis, but due to the lack of proper human cell models, the role of microglia in SCZ is not well studied. We used monozygotic twins discordant for SCZ and healthy individuals to generate human induced pluripotent stem cell-derived microglia to assess the transcriptional and functional differences in microglia between healthy controls, affected twins and unaffected twins. The microglia from affected twins had increased expression of several common inflammation-related genes compared to healthy individuals. Microglia from affected twins had also reduced response to interleukin 1 beta (IL1ß) treatment, but no significant differences in migration or phagocytotic activity. Ingenuity Pathway Analysis (IPA) showed abnormalities related to extracellular matrix signaling. RNA sequencing predicted downregulation of extracellular matrix structure constituent Gene Ontology (GO) terms and hepatic fibrosis pathway activation that were shared by microglia of both affected and unaffected twins, but the upregulation of major histocompatibility complex (MHC) class II receptors was observed only in affected twin microglia. Also, the microglia of affected twins had heterogeneous response to clozapine, minocycline, and sulforaphane treatments. Overall, despite the increased expression of inflammatory genes, we observed no clear functional signs of hyperactivation in microglia from patients with SCZ. We conclude that microglia of the patients with SCZ have gene expression aberrations related to inflammation response and extracellular matrix without contributing to increased microglial activation.

ER and SOCE Ca2+ signals are not required for directed cell migration in human microglia.

The central nervous system (CNS) is constantly surveilled by microglia, highly motile and dynamic cells deputed to act as the first line of immune defense in the brain and spinal cord. Alterations in the homeostasis of the CNS are detected by microglia that respond by migrating toward the affected area. Understanding the mechanisms controlling directed cell migration of microglia is crucial to dissect their responses to neuroinflammation and injury. We used a combination of pharmacological and genetic approaches to explore the involvement of calcium (Ca2+) signaling in the directed migration of induced pluripotent stem cell (iPSC)-derived microglia challenged with a purinergic stimulus. This approach mimics cues originating from injury of the CNS. Unexpectedly, simultaneous imaging of microglia migration and intracellular Ca2+ changes revealed that this phenomenon does not require Ca2+ signals generated from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) pathways. Instead, we find evidence that human microglial chemotaxis to purinergic signals is mediated by cyclic AMP in a Ca2+-independent manner. These results challenge prevailing notions, with important implications in neurological conditions characterized by perturbation in Ca2+ homeostasis.

Microglial APOE4: more is less and less is more.

Apolipoprotein E (APOE) is the single greatest genetic risk factor for late onset Alzheimer's disease (AD). Yet, the cell-specific effects of APOE on microglia function have remained unclear. Fortunately, two comprehensive new studies published in the latest issue of Nature Immunology have employed complementary gain-of-function and loss-of-function approaches to provide critical new insight into the impact of microglial APOE on AD pathogenesis.

Microglia promote anti-tumour immunity and suppress breast cancer brain metastasis.

Breast cancer brain metastasis (BCBM) is a lethal disease with no effective treatments. Prior work has shown that brain cancers and metastases are densely infiltrated with anti-inflammatory, protumourigenic tumour-associated macrophages, but the role of brain-resident microglia remains controversial because they are challenging to discriminate from other tumour-associated macrophages. Using single-cell RNA sequencing, genetic and humanized mouse models, we specifically identify microglia and find that they play a distinct pro-inflammatory and tumour-suppressive role in BCBM. Animals lacking microglia show increased metastasis, decreased survival and reduced natural killer and T cell responses, showing that microglia are critical to promote anti-tumour immunity to suppress BCBM. We find that the pro-inflammatory response is conserved in human microglia, and markers of their response are associated with better prognosis in patients with BCBM. These findings establish an important role for microglia in anti-tumour immunity and highlight them as a potential immunotherapy target for brain metastasis.

Human microglia maturation is underpinned by specific gene regulatory networks.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage- and disease-specific phenotypes.

Alteration of microglial metabolism and inflammatory profile contributes to neurotoxicity in a hiPSC-derived microglia model of frontotemporal dementia 3.

Frontotemporal dementia (FTD) is a common cause of early-onset dementia, with no current treatment options. FTD linked to chromosome 3 (FTD3) is a rare sub-form of the disease, caused by a point mutation in the Charged Multivesicular Body Protein 2B (CHMP2B). This mutation causes neuronal phenotypes, such as mitochondrial deficiencies, accompanied by metabolic changes and interrupted endosomal-lysosomal fusion. However, the contribution of glial cells to FTD3 pathogenesis has, until recently, been largely unexplored. Glial cells play an important role in most neurodegenerative disorders as drivers and facilitators of neuroinflammation. Microglia are at the center of current investigations as potential pro-inflammatory drivers. While gliosis has been observed in FTD3 patient brains, it has not yet been systematically analyzed. In the light of this, we investigated the role of microglia in FTD3 by implementing human induced pluripotent stem cells (hiPSC) with either a heterozygous or homozygous CHMP2B mutation, introduced into a healthy control hiPSC line via CRISPR-Cas9 precision gene editing. These hiPSC were differentiated into microglia to evaluate the pro-inflammatory profile and metabolic state. Moreover, hiPSC-derived neurons were cultured with conditioned microglia media to investigate disease specific interactions between the two cell populations. Interestingly, we identified two divergent inflammatory microglial phenotypes resulting from the underlying mutations: a severe pro-inflammatory profile in CHMP2B homozygous FTD3 microglia, and an "unresponsive" CHMP2B heterozygous FTD3 microglial state. These findings correlate with our observations of increased phagocytic activity in CHMP2B homozygous, and impaired protein degradation in CHMP2B heterozygous FTD3 microglia. Metabolic mapping confirmed these differences, revealing a metabolic reprogramming of the CHMP2B FTD3 microglia, displayed as a compensatory up-regulation of glutamine metabolism in the CHMP2B homozygous FTD3 microglia. Intriguingly, conditioned CHMP2B homozygous FTD3 microglia media caused neurotoxic effects, which was not evident for the heterozygous microglia. Strikingly, IFN-γ treatment initiated an immune boost of the CHMP2B heterozygous FTD3 microglia, and conditioned microglia media exposure promoted neural outgrowth. Our findings indicate that the microglial profile, activity, and behavior is highly dependent on the status of the CHMP2B mutation. Our results suggest that the heterozygous state of the mutation in FTD3 patients could potentially be exploited in form of immune-boosting intervention strategies to counteract neurodegeneration.

Mitochondria dysregulation contributes to secondary neurodegeneration progression post-contusion injury in human 3D in vitro triculture brain tissue model.

Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury composed of neurons, microglia, and astrocytes and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for developing therapies for TBI patients.

Astrocytic response mediated by the CLU risk allele inhibits OPC proliferation and myelination in a human iPSC model.

The C allele of rs11136000 variant in the clusterin (CLU) gene represents the third strongest known genetic risk factor for late-onset Alzheimer's disease. However, whether this single-nucleotide polymorphism (SNP) is functional and what the underlying mechanisms are remain unclear. In this study, the CLU rs11136000 SNP is identified as a functional variant by a small-scale CRISPR-Cas9 screen. Astrocytes derived from isogenic induced pluripotent stem cells (iPSCs) carrying the "C" or "T" allele of the CLU rs11136000 SNP exhibit different CLU expression levels. TAR DNA-binding protein-43 (TDP-43) preferentially binds to the "C" allele to promote CLU expression and exacerbate inflammation. The interferon response and CXCL10 expression are elevated in cytokine-treated C/C astrocytes, leading to inhibition of oligodendrocyte progenitor cell (OPC) proliferation and myelination. Accordingly, elevated CLU and CXCL10 but reduced myelin basic protein (MBP) expression are detected in human brains of C/C carriers. Our study uncovers a mechanism underlying reduced white matter integrity observed in the CLU rs11136000 risk "C" allele carriers.

CRISPR generation of CSF1R-G795A human microglia for robust microglia replacement in a chimeric mouse model.

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.

Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro.

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer's disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

Moderate intrinsic phenotypic alterations in C9orf72 ALS/FTD iPSC-microglia despite the presence of C9orf72 pathological features.

While motor and cortical neurons are affected in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), it remains largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We differentiated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels, and generate dipeptide repeat proteins. Healthy control and C9orf72 ALS/FTD iPSC-MG equally express microglial specific genes and perform microglial functions, including inflammatory cytokine release and phagocytosis of extracellular cargos, such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. RNA sequencing analysis revealed select transcriptional changes of genes associated with neuroinflammation or neurodegeneration in diseased microglia yet no significant differentially expressed microglial-enriched genes. Moderate molecular and functional differences were observed in C9orf72 iPSC-MG mono-cultures despite the presence of C9orf72 pathological features suggesting that a diseased microenvironment may be required to induce phenotypic changes in microglial cells and the associated neuronal dysfunction seen in C9orf72 ALS/FTD neurodegeneration.

Novel Vaccine against Pathological Pyroglutamate-Modified Amyloid Beta for Prevention of Alzheimer's Disease.

Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE3Aß) is a highly pathogenic molecule with increased neurotoxicity and propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE3Aß represents a major constituent of the amyloid plaque. The data show that pE3Aß 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 pE3Aß 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 pE3Aß3-11 fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in AdvaxCpG adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 105-106 against pE3Aß and 103-104 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.

Single-nuclei transcriptome analysis of Huntington disease iPSC and mouse astrocytes implicates maturation and functional deficits.

Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene that alters cellular homeostasis, particularly in the striatum and cortex. Astrocyte signaling that establishes and maintains neuronal functions are often altered under pathological conditions. We performed single-nuclei RNA-sequencing on human HD patient-induced pluripotent stem cell (iPSC)-derived astrocytes and on striatal and cortical tissue from R6/2 HD mice to investigate high-resolution HD astrocyte cell state transitions. We observed altered maturation and glutamate signaling in HD human and mouse astrocytes. Human HD astrocytes also showed upregulated actin-mediated signaling, suggesting that some states may be cell-autonomous and human specific. In both species, astrogliogenesis transcription factors may drive HD astrocyte maturation deficits, which are supported by rescued climbing deficits in HD drosophila with NFIA knockdown. Thus, dysregulated HD astrocyte states may induce dysfunctional astrocytic properties, in part due to maturation deficits influenced by astrogliogenesis transcription factor dysregulation.

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Microglia states and nomenclature: A field at its crossroads.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

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.