Microglia dynamically support brain health through the induction of specialized activation states in response to injury or disease. Activation of the interferon-responsive microglia (IRM) state has been identified across neurodevelopmental windows, age-related cognitive decline, and neurodegenerative diseases. Functionally, IRM have been linked to synaptic pruning, dead cell removal, and neuroinflammation, making this state critical to brain homeostasis. While the functional importance of this state is becoming increasingly clear, our understanding of the regulatory networks that govern IRM induction remain incomplete. To systematically identify genetic regulators of the IRM state, we conducted a genome-wide CRISPR interference screen in human iPSC-derived microglia using IFIT1 as a representative IRM marker. We identified 772 genes that modulate IRM, including canonical type I interferon signaling genes (IFNAR2, TYK2, STAT1/2, USP18) and newly described regulators. We uncovered a non-canonical role for the CCR4-NOT transcription complex subunit 10, CNOT10, in IRM activation. This work provides a comprehensive resource that can be applied to dissect the functions of interferon-responsive microglia and highlights both established and novel targets for modulating microglial interferon signaling in health and disease.

Transgenes are often silenced upon differentiation of pluripotent stem cells using conventional expression systems. Here, we developed the TK4 PiggyBac vector to conduct a comparative analysis to evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of CAG and Ubc promoters with the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) can prevent transgene silencing during differentiation, whereas chromatin insulators have less impact on sustained expression. Three novel safe harbor loci, distant from known genes, as well as the citrate lyase beta-like (CLYBL) locus, similarly support the prevention of transgene silencing. Remarkably, the TK4 vector showed complete resistance to silencing across various neuronal and microglial differentiation protocols, as independently confirmed by seven laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation and holds potential for broad applications in various research fields.

Altered microglial lipid metabolism is heavily implicated in Alzheimer’s disease (AD) and aging. Recently, protocols were developed to generate human induced pluripotent stem cell-derived microglia-like cells (iMGL) to study microglial function in vitro, including embryoid body-based methods and induced transcription factor (iTF)-dependent approaches. Here, we performed comparative lipidomics on iMGL from these methods and report major differences in multiple lipid classes, including triglycerides (TGs), a storage form of fatty acids implicated in microglial reactivity. TGs are strongly increased in iTF microglia due to the absence of a media supplement (B-27). Supplementing iTF microglia with B-27, or its component L-carnitine, reduces TGs and promotes a homeostatic state. B-27 also renders iTF microglia metabolically responsive to immune stimuli. Overall, our data show that iMGL differentiation methods have a major impact on microglial lipidomes and warrant attention when studying AD and neuroinflammatory processes involving lipids.

Microglia, the brain’s innate immune cells, can adopt a wide variety of activation states relevant to health and disease. Dysregulation of microglial activation occurs in numerous brain disorders, and driving or inhibiting specific states could be therapeutic. To discover regulators of microglial activation states, we conducted CRISPR interference screens in induced pluripotent stem cell (iPSC)-derived microglia for inhibitors and activators of six microglial states. We characterized 31 regulators at the single-cell transcriptomic and cell-surface proteome level in two distinct iPSC-derived microglia models, uncovering protein markers of relevant states. We functionally characterized several multi-state regulators. ZNF532 and PRDM1 knockdown drive disease-associated, lipid-rich signatures and enhance phagocytosis while showing opposing effects on antigen-presentation signatures. DNMT1 knockdown results in widespread loss of DNA methylation, activating negative regulators of interferon signaling. These findings provide a framework to direct microglial activation to selectively enrich microglial activation states, define their functional outputs, and inform future therapies.

Many lines of evidence support protective and pathogenic roles of microglia in Alzheimer’s Disease. Microglia associated with amyloid plaques are proposed to acquire a disease-responsive phenotype through sequential Trem2-independent and Trem2-dependent stages. However, the transcriptional mechanisms driving this transition have not been resolved. Here, we intersect the amyloid-responsive epigenomic landscapes of mouse and human microglia with transcriptional and epigenetic responses of macrophages to an in vivo model of sterile inflammation. We provide evidence for a general model in which activation of a common set of regulators, including members of the MITF/TFE and ATF/AP-1 transcription factor families, is required for both Trem2-independent and Trem2-dependent gene expression. Rather than engaging additional transcription factors, Trem2-dependent gene expression results from boosting the activities of selective enhancers that are initially activated during the Trem2-independent stage and are co-bound by MITF/TFE and ATF/AP-1 factors. These findings reveal transcriptional circuitry required for acquisition of disease-responsive microglia phenotypes.

Stem cell-derived extracellular vesicles (EVs) show promise as a therapeutic approach for neurodegenerative diseases, particularly Alzheimer's Disease (AD), where traditional regenerative interventions have achieved limited success. Our previous research demonstrated the neuroprotective benefits of human neural stem cell (hNSC)-derived EVs in 2- and 6-month-old AD mice (5xFAD) that exibited improved cognitive function and reduced AD-related neuropathology. This study aimed to compare the neuroprotective efficacy of EVs derived from two human cell lines: hNSCs from H9 embryonic stem cells and human iPSC-derived microglia (iMGLs). Additionally, we investigated the efficacy of an expanded EV treatment paradigm at subsequently longer time points. Three-month-old 5xFAD mice received weekly retro-orbital vein injections of either hNSC- or iMGL-derived EVs for 4 weeks. Cognitive function testing revealed comparable cognitive improvements in both EV treatment groups compared to vehicle-injected AD mice. Both iMGL- and hNSC-derived EVs significantly reduced amyloid beta plaques, astrogliosis, and microglial activation, while restoring synaptophysin and postsynaptic density protein PSD-95 to control levels in AD brains. Gene expression analysis revealed significantly reduced neuroinflammation and elevated neuroprotective signatures following both EV treatments. MicroRNA analysis of the EV-derived cargo revealed unique and shared miRNA signatures associated with differentially expressed genes in both cell lines. These findings demonstrate the feasibility and neuroprotective benefits of recurrent systemic injections of EVs derived from human NSCs and differentiated human microglia lines in alleviating cognitive dysfunction and neuropathology in Alzheimer's disease.

Microglia dynamically support brain homeostasis through the induction of specialized activation programs or states. One such program is the Interferon-Responsive Microglia state (IRM), which has been identified in developmental windows, aging, and disease. While the functional importance of this state is becoming increasingly clear, our understanding of the regulatory networks that govern IRM induction remain incomplete. To systematically identify genetic regulators of the IRM state, we conducted a genome-wide CRISPR interference (CRISPRi) screen in human iPSC-derived microglia (iPS-Microglia) using IFIT1 as a representative IRM marker. We identified 772 genes that modulate IRM, including canonical type I interferon signaling genes ( IFNAR2, TYK2, STAT1/2, USP18 ) and novel regulators. We uncovered a non-canonical role for the CCR4-NOT complex subunit CNOT10 in IRM activation, independent of its traditional function. This work provides a comprehensive resource for dissecting IRM biology and highlights both established and novel targets for modulating microglial interferon signaling in health and disease. ### Competing Interest Statement MK is a co-scientific founder of Montara Therapeutics and serves on the Scientific Advisory Boards of Montara Therapeutics, Engine Biosciences, Alector and Neurocrine, and is an advisor to Modulo Bio and Theseus Therapies. MK is an inventor on US Patent 11,254,933 related to CRISPRi and CRISPRa screening, and on a US Patent application on in vivo screening methods. Alzheimer’s Association, AARF-22-973222, ZEN-22-969903, ADSF-21-831212-C Larry L. Hillblom Foundation, 2022-A-016-FEL National Science Foundation, 2034836 Chan Zuckerberg Initiative, CP2-1-0000000332

Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified ADNP, a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.

INTRODUCTION: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. METHODS: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. RESULTS: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. DISCUSSION: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.

Large-scale genetic studies and eQTL analyses have revealed microglia as critical players in Alzheimer's disease (AD). Consequently, there has been a deep focus on defining microglial activation states across models of AD. These studies have revealed several activation states to be enriched in AD including Interferon-Responsive Microglia (IRM). IRM are hypothesized to represent a toxic activation state that promotes chronic neuroinflammation and loss of synapses. Thus, negative regulators of the IRM response are likely to be beneficial therapeutic agents to target microglial activation in AD. To uncover regulators of this maladaptive interferon-response state, we performed a genome-wide CRISPR interference screen for IFIT1 expression, a conserved marker of the interferon-response state. Screening was performed in human iPSC-derived microglia pre-stimulated with IFNβ to enrich discovery of negative regulators of IRM which are proposed to slow tau accumulation and synaptic loss in AD. These experiments uncovered both canonical regulators of interferon signaling as well as novel regulators of the IRM state including RNA processing and DNA methylation pathways. Importantly, these inhibitory effects were specific to regulation of the IRM state and did not broadly impair activation towards more beneficial disease response states. Further investigation into the mechanisms that underlie inhibition of interferon-response have converged on nucleic acid sensing as a critically important regulator of sterile IRM. Mounting evidence shows that accumulation of cytoplasmic nucleic acids occurs as an early pathogenic marker of AD and that altering microglial responses to these disease-associated molecular patterns can abrogate symptom onset in murine models. We have discovered novel regulators of nucleic acid sensing and the interferon-responsive state using iPSC-derived microglial models and CRISPR screening technologies. These findings provide a foundation to support development of targeted therapies that inhibit IRM in vivo and restore protective functions of microglia.

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 (IL1β) 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.

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 extending their processes or - following major injuries - 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 human 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.

Advanced ScienceVolume 11, Issue 20 2470114 Inside Front CoverOpen Access Neurotoxic Microglial Activation via IFNγ-Induced Nrf2 Reduction Exacerbating Alzheimer's Disease (Adv. Sci. 20/2024) You Jung Kang, You Jung KangSearch for more papers by this authorSeung Jae Hyeon, Seung Jae HyeonSearch for more papers by this authorAmanda McQuade, Amanda McQuadeSearch for more papers by this authorJiwoon Lim, Jiwoon LimSearch for more papers by this authorSeung Hyun Baek, Seung Hyun BaekSearch for more papers by this authorYen N. Diep, Yen N. DiepSearch for more papers by this authorKhanh V. Do, Khanh V. DoSearch for more papers by this authorYeji Jeon, Yeji JeonSearch for more papers by this authorDong-Gyu Jo, Dong-Gyu JoSearch for more papers by this authorC. Justin Lee, C. Justin LeeSearch for more papers by this authorMathew Blurton-Jones, Mathew Blurton-JonesSearch for more papers by this authorHoon Ryu, Hoon RyuSearch for more papers by this authorHansang Cho, Hansang ChoSearch for more papers by this author You Jung Kang, You Jung KangSearch for more papers by this authorSeung Jae Hyeon, Seung Jae HyeonSearch for more papers by this authorAmanda McQuade, Amanda McQuadeSearch for more papers by this authorJiwoon Lim, Jiwoon LimSearch for more papers by this authorSeung Hyun Baek, Seung Hyun BaekSearch for more papers by this authorYen N. Diep, Yen N. DiepSearch for more papers by this authorKhanh V. Do, Khanh V. DoSearch for more papers by this authorYeji Jeon, Yeji JeonSearch for more papers by this authorDong-Gyu Jo, Dong-Gyu JoSearch for more papers by this authorC. Justin Lee, C. Justin LeeSearch for more papers by this authorMathew Blurton-Jones, Mathew Blurton-JonesSearch for more papers by this authorHoon Ryu, Hoon RyuSearch for more papers by this authorHansang Cho, Hansang ChoSearch for more papers by this author First published: 28 May 2024 https://doi.org/10.1002/advs.202470114AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract Alzheimer's Diseases In article number 2304357, Hansang Cho and co-workers investigate neurotoxic microgliosis in Alzheimer's disease (AD). This study reveals that AD neuron-reactive astrocyte interaction induces production of key cytokine (IFNγ) and accumulation of oxidative stress (H2O2), which activate proinflammatory microglia. The proinflammatory microglia in tern exacerbate synaptic impairment, phosphorylated-tau accumulation, and neuronal loss at the end. The cover was illustrated by You Jung Kang. Volume11, Issue20May 28, 20242470114 RelatedInformation

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 (Aβ)-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.

Human genetic studies and quantitative trait loci (eQTL) analyses have implicated microglia, the innate immune cells of the brain, as critical players in Alzheimer’s disease (AD). In response, the field has focused on defining microglial transcriptional states across many models of neurodegeneration. Integrating these studies, we find several microglial activation states commonly enriched in AD. These include Disease Associated Microglia, Antigen-Presenting Microglia, and Interferon-Responsive Microglia. To truly make use of this data, we now must move towards understanding the functional consequence of these microglial activation states and uncovering the mechanisms that drive changes in cell states during disease. To determine regulators of the interferon-responsive microglial activation state, we performed a functional genomic screen using CRISPR interference (CRISPRi). To avoid bottlenecking of CRISPR guide RNAs and accommodate the large numbers of cells needed for screening, we implemented a novel transcription-factor driven approach to differentiate iPSC-derived microglia. After differentiation, microglia harboring individual gene knockdowns were primed with type I interferon and screened on expression of IFIT1, a key marker of the interferon-responsive microglial state. These experiments have uncovered both canonical regulators of interferon signaling as well as novel regulators of the interferon-responsive microglial state. Furthermore, several known AD risk genes were shown to inhibit microglial entry into this state, suggesting a potential mechanism whereby these nodes may influence disease risk. Recent transcriptomic studies have uncovered significant heterogeneity of microglial states including the discovery of several disease associated states. However, it remains unclear whether these states represent beneficial, disease-fighting functions or detrimental, disease-promoting functions. Gaining the ability to selectively control entry into specific microglial states will allow novel investigations into how these specific microglial states interact with and guide disease trajectories.

ApoE4 is the major genetic risk factor for Alzheimer’s disease and the main lipid transporter in brain. How ApoE4 drives AD pathogenesis, and how this is related to changes in lipid metabolism, is not well understood. This lack of mechanistic insight has hampered the development of interventions that can reverse the pathological effects of ApoE4. Here we perform integrated multiomic analysis (lipidomics, metabolomics, transcriptomics and proteomics) on isogenic iPSC-derived glia carrying an ApoE4/4, ApoE3/3 or ApoE-knockout genotype. We employ pathway enrichment to identify candidate biological processes that contribute to AD pathogenesis and perform a number of biochemical- and intervention experiments to gain insight into the biological mechanism. We feed iPSC-derived glia with different lipid species to better understand the interaction between lipids and immune dysfunction. Lastly, we generate a new open-access resource (the Neurolipid atlas), to allow others in the field to interact with our lipid data. Our lipidomic analysis shows that ApoE4 has a major effect on lipid metabolism in human iPSC-derived astrocytes and microglia. In both astrocytes and microglia, ApoE4 causes accumulation of cholesteryl esters (CE), triglycerides (TAG) as well changes in ceramide metabolism. Interestingly, transcriptomic and proteomic analysis in both cell types shows that ApoE4 glia have strongly upregulated cholesterol synthesis despite having already high levels of stored cholesterol (CE). Astrocytes and microglia show very distinct adaptations to ApoE4 in terms of energy production. Whereas ApoE4 astrocytes have higher levels of mitochondrial proteins, ApoE4 microglia downregulate mitochondrial proteins but have increased levels of key glycolytic proteins. Immune profiles, including DAM signatures in microglia, are changed in a similar direction in both ApoE4 astrocytes and microglia. Lipid feeding also directly affect these immune pathways. Our data indicate that astrocytes and microglia display both common and distinct immunometabolic adaptations to the ApoE4 genotype. Interestingly, both ApoE4 microglia and astrocytes have increased stored cholesterol yet keep upregulating cholesterol synthesis, indicating a possibly pathogenic broken feedback loop. We also show that this altered lipid metabolism is tightly coupled to immune dysfunction and propose novel mechanisms and interventions that may normalize ApoE4 dysfunction in human glia in AD.

The discovery of TREM2 as a myeloid-speci fi c 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 pro fi le microglia differentiated from isogenic, CRISPR-modi fi ed TREM2-knockout induced pluripotent stem cell (iPSC) lines. By combining transcriptomic and functional analyses with a chimeric AD mouse model, we fi nd that TREM2 deletion reduces microglial survival, impairs phagocytosis of key substrates including APOE, and inhibits SDF-1 α /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 fl ow cytometry and immunohistochemistry. Taken together, these studies reveal both conserved and novel aspects of human TREM2 biology that likely play critical roles

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.

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.