Kuti Baruch

A Unique Microglia Type Associated with Restricting Development of Alzheimer’s Disease

Alzheimers disease (AD) is a detrimental neurodegenerative disease with no effective treatments. Due to cellular heterogeneity, defining the roles of immune cell subsets in AD onset and progression has been challenging. Using transcriptional single-cell sorting, we comprehensively map all immune populations in wild-type and AD-transgenic (Tg-AD) mouse brains. We describe a novel microglia type associated with neurodegenerative diseases (DAM) and identify markers, spatial localization, and pathways associated with these cells. Immunohistochemical staining of mice and human brain slices shows DAM with intracellular/phagocytic Aβ particles. Single-cell analysis of DAM in Tg-AD and triggering receptor expressed on myeloid cells 2 (Trem2)-/- Tg-AD reveals that the DAM program is activated in a two-step process. Activation is initiated in a Trem2-independent manner that involves downregulation of microglia checkpoints, followed by activation of a Trem2-dependent program. This unique microglia-type has the potential to restrict neurodegeneration, which may have important implications for future treatment of AD and other neurodegenerative diseases. VIDEO ABSTRACT.

Microglia development follows a stepwise program to regulate brain homeostasis.

Microglia development follows a stepwise program Microglia are cells that defend the central nervous system. However, because they migrate into the brain during development, the changes that they undergo, including those that affect gene expression, have been difficult to document. Matcovitch-Natan et al. transcriptionally profiled gene expression and analyzed epigenetic signatures of microglia at the single-cell level in the early postnatal life of mice. They identified three stages of microglia development, which are characterized by gene expression and linked with chromatin changes, occurring in sync with the developing brain. Furthermore, they showed that the proper development of microglia is affected by the microbiome. Science, this issue p. 789 The microbiota help regulate the development of active immune defense in the central nervous system of mice. INTRODUCTION Microglia, as the resident myeloid cells of the central nervous system, play an important role in life-long brain maintenance and in pathology. Microglia are derived from erythromyeloid progenitors that migrate to the brain starting at embryonic day 8.5 and continuing until the blood-brain barrier is formed; after this, self-renewal is the only source of new microglia in the healthy brain. As the brain develops, microglia must perform different functions to accommodate temporally changing needs: first, actively engaging in synapse pruning and neurogenesis, and later, maintaining homeostasis. Although the interactions of microglia with the brain environment at steady state and in response to immune challenges have been well studied, their dynamics during development have not been fully elucidated. RATIONALE We systematically studied the transcriptional and epigenomic regulation of microglia throughout brain development to decipher the dynamics of the chromatin state and gene networks governing the transformation from yolk sac progenitor to adult microglia. We used environmental and genetic perturbation models to investigate how timed disruptions to microglia impact their natural development. RESULTS Global profiles of transcriptional states indicated that microglia development proceeds through three distinct temporal stages, which we define as early microglia (until embryonic day 14), pre-microglia (from embryonic day 14 to a few weeks after birth), and adult microglia (from a few weeks after birth onward). ATAC-seq (assay for transposase-accessible chromatin followed by sequencing) for chromatin accessibility and ChIP-seq (chromatin immunoprecipitation followed by sequencing) for histone modifications further characterized the differential regulatory elements in each developmental phase. Single-cell transcriptome analysis revealed minor mixing of the gene expression programs across phases, suggesting that individual cells shift their regulatory networks during development in a coordinated manner. Specific markers and regulatory factors distinguish each phase: For example, we identified MAFB as an important transcription factor of the adult microglia program. Microglia-specific knockout of MafB led to disruption of homeostasis in adulthood and increased expression of interferon and inflammation pathways. We found that microglia from germ-free mice exhibited dysregulation of dozens of genes associated with the adult phase and immune response. In addition, maternal immune activation, which has been linked to behavioral disorders in adult offspring, had the greatest impact on pre-microglia, resulting in a transcriptional shift toward the more advanced developmental stage. CONCLUSION Our work identifies a stepwise developmental program of microglia in synchrony with the developing brain. Each stage of microglia development has evolved distinct pathways for processing the relevant signals from the environment to balance their time-dependent role in neurogenesis with regulation of immune responses that may cause collateral damage. Genetic or environmental perturbations of these pathways can disrupt stage-specific functions of microglia and lead to loss of brain homeostasis, which may be associated with neurodevelopmental disorders. Microglia development proceeds in a stepwise manner. Microglia were isolated from mice throughout development from embryo to adult. Data from population-level RNA-seq, ChIP-seq, and ATAC-seq, as well as single-cell RNA-seq, show that microglia development proceeds through three distinct stages—early, pre-, and adult— with characteristic gene expression and functional states. Perturbations of this developmental process, such as from MafB knockout, lead to disrupted brain homeostasis by the dysregulation of adult and inflammatory genes. Tn5, transposase 5. Microglia, the resident myeloid cells of the central nervous system, play important roles in life-long brain maintenance and in pathology. Despite their importance, their regulatory dynamics during brain development have not been fully elucidated. Using genome-wide chromatin and expression profiling coupled with single-cell transcriptomic analysis throughout development, we found that microglia undergo three temporal stages of development in synchrony with the brain—early, pre-, and adult microglia—which are under distinct regulatory circuits. Knockout of the gene encoding the adult microglia transcription factor MAFB and environmental perturbations, such as those affecting the microbiome or prenatal immune activation, led to disruption of developmental genes and immune response pathways. Together, our work identifies a stepwise microglia developmental program integrating immune response pathways that may be associated with several neurodevelopmental disorders.

Aging-induced type I interferon response at the choroid plexus negatively affects brain function

Excess signaling is bad for the aging brain Preventing antiviral-like responses may protect function in the aging brain. Baruch et al. monitored messenger RNA production in the choroid plexus, the interface between the blood and cerebrospinal fluid, in young and old mice (see the Perspective by Ransohoff). They detected an inflammatory response in older mice not present in the brain of young mice that was also seen in old aged human samples postmortem. Preventing signaling by the cytokine interferon-I, which normally helps in the antiviral response of the immune system, helped prevent the decrease in cognitive function seen in aged mice. Science, this issue p. 89; see also p. 36 Excess signaling by type I interferon contributes to cognitive decline in aged mice. [Also see Perspective by Ransohoff] Aging-associated cognitive decline is affected by factors produced inside and outside the brain. By using multiorgan genome-wide analysis of aged mice, we found that the choroid plexus, an interface between the brain and the circulation, shows a type I interferon (IFN-I)–dependent gene expression profile that was also found in aged human brains. In aged mice, this response was induced by brain-derived signals, present in the cerebrospinal fluid. Blocking IFN-I signaling within the aged brain partially restored cognitive function and hippocampal neurogenesis and reestablished IFN-II–dependent choroid plexus activity, which is lost in aging. Our data identify a chronic aging-induced IFN-I signature, often associated with antiviral response, at the brain’s choroid plexus and demonstrate its negative influence on brain function, thereby suggesting a target for ameliorating cognitive decline in aging.

The resolution of neuroinflammation in neurodegeneration: leukocyte recruitment via the choroid plexus

Inflammation is an integral part of the bodys physiological repair mechanism, unless it remains unresolved and becomes pathological, as evident in the progressive nature of neurodegeneration. Based on studies from outside the central nervous system (CNS), it is now understood that the resolution of inflammation is an active process, which is dependent on well‐orchestrated innate and adaptive immune responses. Due to the immunologically privileged status of the CNS, such resolution mechanism has been mostly ignored. Here, we discuss resolution of neuroinflammation as a process that depends on a network of immune cells operating in a tightly regulated sequence, involving the brains choroid plexus (CP), a unique neuro‐immunological interface, positioned to integrate signals it receives from the CNS parenchyma with signals coming from circulating immune cells, and to function as an on‐alert gate for selective recruitment of inflammation‐resolving leukocytes to the inflamed CNS parenchyma. Finally, we propose that functional dysregulation of the CP reflects a common underlying mechanism in the pathophysiology of neurodegenerative diseases, and can thus serve as a potential novel target for therapy.

CNS-specific immunity at the choroid plexus shifts toward destructive Th2 inflammation in brain aging

The adaptive arm of the immune system has been suggested as an important factor in brain function. However, given the fact that interactions of neurons or glial cells with T lymphocytes rarely occur within the healthy CNS parenchyma, the underlying mechanism is still a mystery. Here we found that at the interface between the brain and blood circulation, the epithelial layers of the choroid plexus (CP) are constitutively populated with CD4+ effector memory cells with a T-cell receptor repertoire specific to CNS antigens. With age, whereas CNS specificity in this compartment was largely maintained, the cytokine balance shifted in favor of the T helper type 2 (Th2) response; the Th2-derived cytokine IL-4 was elevated in the CP of old mice, relative to IFN-γ, which decreased. We found this local cytokine shift to critically affect the CP epithelium, triggering it to produce the chemokine CCL11 shown to be associated with cognitive dysfunction. Partial restoration of cognitive ability in aged mice, by lymphopenia-induced homeostasis-driven proliferation of memory T cells, was correlated with restoration of the IL-4:IFN-γ ratio at the CP and modulated the expression of plasticity-related genes at the hippocampus. Our data indicate that the cytokine milieu at the CP epithelium is affected by peripheral immunosenescence, with detrimental consequences to the aged brain. Amenable to immunomodulation, this interface is a unique target for arresting age-related cognitive decline.

IFN-γ-dependent activation of the brain’s choroid plexus for CNS immune surveillance and repair

Infiltrating T cells and monocyte-derived macrophages support central nervous system repair. Although infiltration of leucocytes to the injured central nervous system has recently been shown to be orchestrated by the brains choroid plexus, the immunological mechanism that maintains this barrier and regulates its activity as a selective gate is poorly understood. Here, we hypothesized that CD4(+) effector memory T cells, recently shown to reside at the choroid plexus stroma, regulate leucocyte trafficking through this portal through their interactions with the choroid plexus epithelium. We found that the naïve choroid plexus is populated by T helper 1, T helper 2 and regulatory T cells, but not by encephalitogenic T cells. In vitro findings revealed that the expression of immune cell trafficking determinants by the choroid plexus epithelium is specifically induced by interferon-γ. Tumour necrosis factor-α and interferon-γ reciprocally controlled the expression of their receptors by the choroid plexus epithelium, and had a synergistic effect in inducing the epithelial expression of trafficking molecules. In vivo, interferon-γ-dependent signalling controlled trafficking through the choroid plexus; interferon-γ receptor knockout mice exhibited reduced levels of T cells and monocyte entry to the cerebrospinal fluid and impaired recovery following spinal cord injury. Moreover, reduced expression of trafficking molecules by the choroid plexus was correlated with reduced CD4(+) T cells in the choroid plexus and cerebrospinal fluid of interferon-γ receptor knockout mice. Similar effect on the expression of trafficking molecules by the choroid plexus was found in bone-marrow chimeric mice lacking interferon-γ receptor in the central nervous system, or reciprocally, lacking interferon-γ in the circulation. Collectively, our findings attribute a novel immunological plasticity to the choroid plexus epithelium, allowing it to serve, through interferon-γ signalling, as a tightly regulated entry gate into the central nervous system for circulating leucocytes immune surveillance under physiological conditions, and for repair following acute injury.

Breaking immune tolerance by targeting Foxp3(+) regulatory T cells mitigates Alzheimer's disease pathology.

Alzheimers disease (AD) is a neurodegenerative disorder in which chronic neuroinflammation contributes to disease escalation. Nevertheless, while immunosuppressive drugs have repeatedly failed in treating this disease, recruitment of myeloid cells to the CNS was shown to play a reparative role in animal models. Here we show, using the 5XFAD AD mouse model, that transient depletion of Foxp3+ regulatory T cells (Tregs), or pharmacological inhibition of their activity, is followed by amyloid-β plaque clearance, mitigation of the neuroinflammatory response and reversal of cognitive decline. We further show that transient Treg depletion affects the brains choroid plexus, a selective gateway for immune cell trafficking to the CNS, and is associated with subsequent recruitment of immunoregulatory cells, including monocyte-derived macrophages and Tregs, to cerebral sites of plaque pathology. Our findings suggest targeting Treg-mediated systemic immunosuppression for treating AD.

PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease

Systemic immune suppression may curtail the ability to mount the protective, cell-mediated immune responses that are needed for brain repair. By using mouse models of Alzheimers disease (AD), we show that immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway evokes an interferon (IFN)-γ–dependent systemic immune response, which is followed by the recruitment of monocyte-derived macrophages to the brain. When induced in mice with established pathology, this immunological response leads to clearance of cerebral amyloid-β (Aβ) plaques and improved cognitive performance. Repeated treatment sessions were required to maintain a long-lasting beneficial effect on disease pathology. These findings suggest that immune checkpoints may be targeted therapeutically in AD.

CNS-specific T cells shape brain function via the choroid plexus

Adaptive immunity was repeatedly shown to play a role in maintaining lifelong brain function. Under physiological conditions, this activity was associated with CD4+ T cells specific for brain self-antigens. Nevertheless, direct interactions of T cells with the healthy neuronal parenchyma are hardly detectable. Recent studies have identified the brains choroid plexus (CP) as an active neuro-immunological interface, enriched with CNS-specific CD4+ T cells. Strategically positioned for receiving signals from both the central nervous system (CNS) through the cerebrospinal fluid (CSF), and from the circulation through epithelium-immune cell interactions, the CP has recently been recognized as an important immunological compartment in maintaining and restoring brain homeostasis/allostasis. Here, we propose that CNS-specific T cells shape brain function via the CP, and suggest this immunological control to be lost as part of aging, in general, and immune senescence, in particular. Accordingly, the CP may serve as a novel target for immunomodulation to restore brain equilibrium.

Breaking peripheral immune tolerance to CNS antigens in neurodegenerative diseases: Boosting autoimmunity to fight-off chronic neuroinflammation

Immune cell infiltration to the brains territory was considered for decades to reflect a pathological process in which immune cells attack the central nervous system (CNS); such a process is observed in the inflammatory autoimmune disease, multiple sclerosis (MS). As neuroinflammatory processes within the CNS parenchyma are also common to other CNS pathologies, regardless of their etiology, including neurodegenerative disorders such as Alzheimers disease (AD) and Amyotrophic lateral sclerosis (ALS), these pathologies have often been compared to MS, a disease that benefits from immunosuppressive therapy. Yet, over the last decade, it became clear that autoimmunity has a bright side, and that it plays a pivotal role in CNS repair following damage. Specifically, autoimmune T cells were found to facilitate CNS healing processes, such as in the case of sterile mechanical injuries to the brain or the spinal cord, mental stress, or biochemical insults. Even more intriguingly, autoimmune T cells were found to be involved in supporting fundamental processes of brain functional integrity, such as in the maintenance of life-long brain plasticity, including spatial learning and memory, and neurogenesis. Importantly, autoimmune T cells are part of a cellular network which, to operate efficiently and safely, requires tight regulation by other immune cell populations, such as regulatory T cells, which are indispensable for maintenance of immunological self-tolerance and homeostasis. Here, we suggest that dysregulation of the balance between peripheral immune suppression, on one hand, and protective autoimmunity, on the other, is an underlying mechanism in the emergence and progression of the neuroinflammatory response associated with chronic neurodegenerative diseases and brain aging. Mitigating chronic neuroinflammation under these conditions necessitates activation, rather than suppression, of the peripheral immune response directed against self. Accordingly, we propose that fighting off acute and chronic neurodegenerative conditions requires breaking peripheral immune tolerance to CNS self-antigens, in order to boost protective autoimmunity. Nevertheless, the optimal approach to fine tune such immune response must be individually explored for each condition.