Author Correction: Novel Hexb-based tools for studying microglia in the CNS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel Hexb-based tools for studying microglia in the CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative and neuroinflammatory diseases of the central nervous system1-4. These highly diverse and specialized functions may be executed by subsets of microglia that already exist in situ, or by specific subsets of microglia that develop from a homogeneous pool of cells on demand. However, little is known about the presence of spatially and temporally restricted subclasses of microglia in the central nervous system during development or disease. Here we combine massively parallel single-cell analysis, single-molecule fluorescence in situ hybridization, advanced immunohistochemistry and computational modelling to comprehensively characterize subclasses of microglia in multiple regions of the central nervous system during development and disease. Single-cell analysis of tissues of the central nervous system during homeostasis in mice revealed specific time- and region-dependent subtypes of microglia. Demyelinating and neurodegenerative diseases evoked context-dependent subtypes of microglia with distinct molecular hallmarks and diverse cellular kinetics. Corresponding clusters of microglia were also identified in healthy human brains, and the brains of patients with multiple sclerosis. Our data provide insights into the endogenous immune system of the central nervous system during development, homeostasis and disease, and may also provide new targets for the treatment of neurodegenerative and neuroinflammatory pathologies.

Author Correction: Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

In this Letter, Dominic Grün and Sagar have been added to the author list (affiliated with Max-Planck-Institute of Immunology and Epigenetics (MPI-IE), Freiburg, Germany). The author list, 'Author contribution' and 'Acknowledgements' sections have been corrected online. See accompanying Amendment.

Effects of tumor treating fields (TTFields) on human mesenchymal stromal cells.

PURPOSE: Mesenchymal stromal cells (MSCs) within the glioblastoma microenvironment have been shown to promote tumor progression. Tumor Treating Fields (TTFields) are alternating electric fields with low intensity and intermediate frequency that exhibit anti-tumorigenic effects. While the effects of TTFields on glioblastoma cells have been studied previously, nothing is known about the influence of TTFields on MSCs. METHODS: Single-cell RNA sequencing and immunofluorescence staining were employed to identify glioblastoma-associated MSCs in patient samples. Proliferation and clonogenic survival of human bone marrow-derived MSCs were assessed after TTFields in vitro. MSC' characteristic surface marker expression was determined using flow cytometry, while multi-lineage differentiation potential was examined with immunohistochemistry. Apoptosis was quantified based on caspase-3 and annexin-V/7-AAD levels in flow cytometry, and senescence was assessed with ß-galactosidase staining. MSCs' migratory potential was evaluated with Boyden chamber assays. RESULTS: Single-cell RNA sequencing and immunofluorescence showed the presence of glioblastoma-associated MSCs in patient samples. TTFields significantly reduced proliferation and clonogenic survival of human bone marrow-derived MSCs by up to 60% and 90%, respectively. While the characteristic surface marker expression and differentiation capacity were intact after TTFields, treatment resulted in increased apoptosis and senescence. Furthermore, TTFields significantly reduced MSCs' migratory capacity. CONCLUSION: We could demonstrate the presence of tumor-associated MSCs in glioblastoma patients, providing a rationale to study the impact of TTFields on MSCs. TTFields considerably increase apoptosis and senescence in MSCs, resulting in impaired survival and migration. The results provide a basis for further analyses on the role of MSCs in glioblastoma patients receiving TTFields.

IGF1R expression by adult oligodendrocytes is not required in the steady-state but supports neuroinflammation.

In the central nervous system (CNS), insulin-like growth factor 1 (IGF-1) regulates myelination by oligodendrocyte (ODC) precursor cells and shows anti-apoptotic properties in neuronal cells in different in vitro and in vivo systems. Previous work also suggests that IGF-1 protects ODCs from cell death and enhances remyelination in models of toxin-induced and autoimmune demyelination. However, since evidence remains controversial, the therapeutic potential of IGF-1 in demyelinating CNS conditions is unclear. To finally shed light on the function of IGF1-signaling for ODCs, we deleted insulin-like growth factor 1 receptor (IGF1R) specifically in mature ODCs of the mouse. We found that ODC survival and myelin status were unaffected by the absence of IGF1R until 15 months of age, indicating that IGF-1 signaling does not play a major role in post-mitotic ODCs during homeostasis. Notably, the absence of IGF1R did neither affect ODC survival nor myelin status upon cuprizone intoxication or induction of experimental autoimmune encephalomyelitis (EAE), models for toxic and autoimmune demyelination, respectively. Surprisingly, however, the absence of IGF1R from ODCs protected against clinical neuroinflammation in the EAE model. Together, our data indicate that IGF-1 signaling is not required for the function and survival of mature ODCs in steady-state and disease.

Neurocognitive and psychiatric post-coronavirus disease 2019 conditions: pathogenic insights of brain dysfunction following severe acute respiratory syndrome coronavirus 2 infection.

PURPOSE OF REVIEW: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), can trigger a myriad of neuropsychiatric manifestations. As a 2-year-old disease (at the writing of this manuscript), its long-term cognitive and neuropsychiatric implications, known as post-COVID-19 conditions, are incompletely recognized and mechanistically obscure. RECENT FINDINGS: Fatigue, anxiety, depression, posttraumatic stress disorder, and cognitive dysfunction are reported more frequently in COVID-19 survivors than in matching, non-COVID-19 population. Risk factors are unclear, including comorbidities, age at COVID-19 onset, or disease severity; women, however, have been reported to be at increased risk than men. Although the frequency of these symptoms decreases over time, at least one in five will have persistent cognitive and neuropsychiatric manifestations one year after recovering from COVID-19. SUMMARY: Neurocognitive and psychiatric post-COVID-19 long-term conditions are frequent and complex multifactorial sequelae. Several acute and chronic factors such as hypoxemia, cerebral thrombotic and inflammatory endothelial damage, and disruption of the blood-brain barrier (leading to parenchymal translocation of pro-inflammatory molecules, cytokines, and cytotoxic T lymphocytes) are involved, leading to microglial activation and astrogliosis. As an evolving topic, evidence derived from prospective studies will expand our understanding of post-COVID-19 these long-term outcomes.

Endogenous retroviruses are associated with hippocampus-based memory impairment.

Retrotransposons compose a staggering 40% of the mammalian genome. Among them, endogenous retroviruses (ERV) represent sequences that closely resemble the proviruses created from exogenous retroviral infection. ERVs make up 8 to 10% of human and mouse genomes and range from evolutionarily ancient sequences to recent acquisitions. Studies in Drosophila have provided a causal link between genomic retroviral elements and cognitive decline; however, in mammals, the role of ERVs in learning and memory remains unclear. Here we studied 2 independent murine models for ERV activation: muMT strain (lacking B cells and antibody production) and intracerebroventricular injection of streptozotocin (ICVI-STZ). We conducted behavioral assessments (contextual fear memory and spatial learning), as well as gene and protein analysis (RNA sequencing, PCR, immunohistochemistry, and western blot assays). Mice lacking mitochondrial antiviral-signaling protein (MAVS) and mice lacking stimulator of IFN genes protein (STING), 2 downstream sensors of ERV activation, provided confirmation of ERV impact. We found that muMT mice and ICVI-STZ mice induced hippocampal ERV activation, as shown by increased gene and protein expression of the Gag sequence of the transposable element intracisternal A-particle. ERV activation was accompanied by significant hippocampus-related memory impairment in both models. Notably, the deficiency of the MAVS pathway was protective against ICVI-STZ-induced cognitive pathology. Overall, our results demonstrate that ERV activation is associated with cognitive impairment in mice. Moreover, they provide a molecular target for strategies aimed at attenuating retroviral element sensing, via MAVS, to treat dementia and neuropsychiatric disorders.

Key role of MIF-related neuroinflammation in neurodegeneration and cognitive impairment in Alzheimer's disease.

BACKGROUND: Macrophage Migration Inhibitory Factor (MIF) is a potent proinflammatory cytokine that promotes the production of other immune mediators. MIF is produced by most cell types in the brain including microglia, astrocytes and neurons. Enhanced expression of MIF might contribute to the persistent activation of glial, chronic neuroinflammation and neurodegeneration. Here, we investigated the effect of MIF on inflammatory markers and spatial learning in a mouse model of sporadic AD and on tau pathology in AD patients. METHODS: We examined the effects of MIF deficiency and pharmacological MIF inhibition in vitro and in vivo. In vitro, quantitative PCR and ELISA were used to assess cytokine production of STZ-treated glial cells. In vivo, C57BL/6 mice were subjected to intracerebroventricular streptozotocin injection (3 mg/kg, ICV-STZ). Neuroinflammation and contextual learning performance were assessed using quantitative PCR and fear conditioning, respectively. Pharmacological MIF inhibition was achieved with intraperitoneal injections of ISO-1 (daily, IP, 20 mg/kg in 5% DMSO in 0.9% NaCl) for 4 weeks following ICV-STZ injection. The findings from ISO-1 treated mice were confirmed in MIF knockout C57BL/6. To assess the role of MIF in human AD, cerebrospinal fluid levels of MIF and hyperphosphorylated tau were measured using ELISA. RESULTS: Administration ICV-STZ resulted in hippocampal dependent cognitive impairment. MIF inhibition with ISO-1 significantly improved the STZ-induced impairment in contextual memory performance, indicating MIF-related inflammation as a major contributor to ICV-STZ-induced memory deficits. Furthermore, inhibition of the MIF resulted in reduced cytokine production in vitro and in vivo. In human subjects with AD at early clinical stages, cerebrospinal fluid levels of MIF were increased in comparison with age-matched controls, and correlated with biomarkers of tau hyper-phosphorylation and neuronal injury hinting at MIF levels as a potential biomarker for early-stage AD. CONCLUSIONS: The present study indicates the key role of MIF in controlling the chronic cytokine release in neuroinflammation related to tau hyperphosphorylation, neurodegeneration, and clinical manifestations of AD, suggesting the potential of MIF inhibition as therapeutic strategy to slow down neurodegeneration and clinical disease progression.

Persistent inflammatory states and their implications in brain disease.

PURPOSE OF REVIEW: Apart from mental, motor and sensory functions, the human central nervous system (CNS) regulates a plethora of homeostatic (autonomic and hormonal) bodily functions. These functions are dependent on specialized neuronal networks. To ensure connectivity of these networks, they are continuously refined and supported by glial cells that outnumber neurons by, according to some accounts, an order of magnitude. Among glial cells, microglia - the brain resident macrophages - plays a crucial role in maintaining neuronal networks. However, in their concomitant role as brain immune cells microglia also engage in inflammatory signaling that may disrupt neuronal networks. Here, we review novel insights for molecular pathways involved in the protective functions of microglia and other immune cells in response to systemic signals and stimuli. RECENT FINDINGS: Recent evidence suggests that aging and systemic disease push individual microglia toward proinflammatory phenotypes compromising the connectivity of neuronal networks, resulting in neuropsychiatric disease. Furthermore, cells (self as well as the microbiome) outside the CNS have been shown to affect neuronal function. SUMMARY: These recent findings have critical implications for mental health, particularly of an aging population, in particular for the development of novel immunomodulatory therapies for brain disease.

Characterization of longitudinal transformation of T2-hyperintensity in oligodendroglioma.

BACKGROUND: Oligodendroglioma (ODG) are CNS resistant tumors characterized by their unique molecular signature, namely a combined deletion of 1p and 19q simultaneously to an IDH1/2 mutation. These tumors have a more favorable clinical outcome compared to other gliomas and a long-time survival that ranges between 10 and 20 years. However, during the course of the disease, multiple recurrences occur and the optimal treatment at each stage of the disease remains unclear. Here we report a retrospective longitudinal observation study of 836 MRI examinations in 44 ODG patients. METHODS: We quantified the volume of T2-hyperintensity to compute growth behavior in dependence of different treatment modalities, using various computational models. RESULTS: The identified growth pattern revealed dynamic changes, which were found to be patient-specific an did not correlate with clinical parameter or therapeutic interventions. Further, we showed that, surgical resection is beneficial for overall survival regardless the WHO grad or timepoint of surgery. To improve overall survival, an extent of resection above 50% is required. Multiple resections do not generally improve overall survival, except a greater extent of resection than in previous surgeries was achieved. CONCLUSIONS: Our data aids to improve the interpretation of MRI images in clinical practice.

Resection of recurrent glioblastoma multiforme in elderly patients: a pseudo-randomized analysis revealed clinical benefit.

INTRODUCTION: Elderly patients constitute an expanding part of our society. Due to a continuously increasing life expectancy, an optimal quality of life is expected even into advanced age. Glioblastoma (GBM) is more common in older patients, but they are still often withheld from efficient treatment due to worry of worse tolerance and have a significantly worse prognosis compared to younger patients. Our retrospective observational study aimed to investigate the therapeutic benefit from a second resection in recurrent glioblastoma of elderly patients. MATERIALS AND METHODS: We included a cohort of 39 elderly patients (> 65 years) with a second resection as treatment option in the case of a tumor recurrence. A causal inference model was built by multiple non- and semiparametric models, which was used to identify matched patients from our elderly GBM database which comprises 538 patients. The matched cohorts were analyzed by a Cox-regression model adjusted by time-dependent covariates. RESULTS: The Cox-regression analysis showed a significant survival benefit (Hazard Ratio: 0.6, 95% CI 0.36-0.9, p-value = 0.0427) for the re-resected group (18.0 months, 95% CI 13.97-23.2 months) compared to the group without re-resection (10.1 months, 95% CI 8.09-20.9 months). No differences in the co-morbidities or hemato-oncological side effects during chemotherapy could be detected. Anesthetic- and surgical complications were rare and comparable to the complication rate of patients undergoing the first-line resection. CONCLUSION: Taken together, in elderly patients, re-resection is an acceptable treatment option in the recurrent state of a glioblastoma. The individual evaluation of the patients' medical status as well as the chances of withstanding general anesthesia needs to be done in close interdisciplinary consultation. If these requirements are met, elderly patients benefit from a re-resection.

The multi-target effects of CNI-1493: convergence of anti-amylodogenic and anti-inflammatory properties in animal models of Alzheimer's disease.

After several decades of Alzheimer's disease (AD) research and failed clinical trials, one can speculate that targeting a single pathway is not sufficient. However, a cocktail of novel therapeutics will constitute a challenging clinical trial. A more plausible approach will capitalize on a drug that has relevant and synergistic multiple-target effects in AD. We have previously demonstrated the efficacy of CNI-1493 in the CRND8 transgenic AD mouse model. Similar to many anti-inflammatory drugs that were tested in preclinical model of AD, it was speculated that the significant effect of CNI-1493 is due to its established anti-inflammatory properties in rodents and humans. In the present study, we set out to elucidate the protective mechanism of CNI-1493 as a drug simultaneously targeting several aspects of AD pathology. Using C1213, a highly similar analogue of CNI-1493 that lacks anti-inflammatory properties, we show that both compounds directly interact with soluble and insoluble Amyloid ß (Aß) aggregates and attenuate Aß cytotoxicity in vitro. Additionally, CNI-1493 and C1213 ameliorated Aß-induced behavioral deficits in nematodes. Finally, C1213 reduced Aß plaque burden and cognitive deficits in transgenic CRND8 mice to a similar extent as previously shown with CNI-1493. Taken together, our findings suggest anti-amyloidogenic activity as a relevant component for the in-vivo efficacy of CNI-1493 and its analogue C1213. Thus, CNI-1493, a drug with proven safety in humans, is a viable candidate for novel multi-target therapeutic approaches to AD.

Rodent models of neuroinflammation for Alzheimer's disease.

Alzheimer's disease remains incurable, and the failures of current disease-modifying strategies for Alzheimer's disease could be attributed to a lack of in vivo models that recapitulate the underlying etiology of late-onset Alzheimer's disease. The etiology of late-onset Alzheimer's disease is not based on mutations related to amyloid-ß (Aß) or tau production which are currently the basis of in vivo models of Alzheimer's disease. It has recently been suggested that mechanisms like chronic neuroinflammation may occur prior to amyloid-ß and tau pathologies in late-onset Alzheimer's disease. The aim of this study is to analyze the characteristics of rodent models of neuroinflammation in late-onset Alzheimer's disease. Our search criteria were based on characteristics of an idealistic disease model that should recapitulate causes, symptoms, and lesions in a chronological order similar to the actual disease. Therefore, a model based on the inflammation hypothesis of late-onset Alzheimer's disease should include the following features: (i) primary chronic neuroinflammation, (ii) manifestations of memory and cognitive impairment, and (iii) late development of tau and Aß pathologies. The following models fit the pre-defined criteria: lipopolysaccharide- and PolyI:C-induced models of immune challenge; streptozotocin-, okadaic acid-, and colchicine neurotoxin-induced neuroinflammation models, as well as interleukin-1ß, anti-nerve growth factor and p25 transgenic models. Among these models, streptozotocin, PolyI:C-induced, and p25 neuroinflammation models are compatible with the inflammation hypothesis of Alzheimer's disease.

Advances in understanding the immunity of the brain and its borders: Focus on brain macrophages.

A recent study outlines the phenotypes of brain border region macrophages in developing, normal and glioblastoma-affected brains. For the first time, the authors show in-vivo turnover of human brain border macrophages. The findings have implications for the understanding of brain border immunity and potential macrophage targeting therapies. KEYPOINTS: Human border region macrophages are distinct from microglia. These distinct phenotypes are established early during embryonal development - Brain border macrophages are partially replaced by bone marrow-derived myeloid cells. The transcriptional phenotypes of glioblastoma-associated macrophage are determined by the anatomical region.

Interaction between subventricular zone microglia and neural stem cells impacts the neurogenic response in a mouse model of cortical ischemic stroke.

After a stroke, the neurogenic response from the subventricular zone (SVZ) to repair the brain is limited. Microglia, as an integral part of the distinctive SVZ microenvironment, control neural stem / precursor cell (NSPC) behavior. Here, we show that discrete stroke-associated SVZ microglial clusters negatively impact the innate neurogenic response, and we propose a repository of relevant microglia-NSPC ligand-receptor pairs. After photothrombosis, a mouse model of ischemic stroke, the altered SVZ niche environment leads to immediate activation of microglia in the niche and an abnormal neurogenic response, with cell-cycle arrest of neural stem cells and neuroblast cell death. Pharmacological restoration of the niche environment increases the SVZ-derived neurogenic repair and microglial depletion increases the formation and survival of newborn neuroblasts in the SVZ. Therefore, we propose that altered cross-communication between microglial subclusters and NSPCs regulates the extent of the innate neurogenic repair response in the SVZ after stroke.