Scalable, cell type-selective, AAV-based in vivo CRISPR screening in the mouse brain.

CRISPR-based genetic screening directly in mammalian tissues in vivo is challenging due to the need for scalable, cell-type selective delivery and recovery of guide RNA libraries. We developed an in vivo adeno-associated virus-based and Cre recombinase-dependent workflow for cell type-selective CRISPR interference screening in mouse tissues. We demonstrate the power of this approach by identifying neuron-essential genes in the mouse brain using a library targeting over 2000 genes.

CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.

The sheer complexity of the brain has complicated our ability to understand its cellular mechanisms in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3ß inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE-ε4, a risk variant for Alzheimer's Disease, in its effect on neuronal survival. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

Reactive astrocytes transduce inflammation in a blood-brain barrier model through a TNF-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin.

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function. Pathological transformation of astrocytes to reactive states can be protective or harmful to BBB function. Here, using a human induced pluripotent stem cell (iPSC)-derived BBB co-culture model, we show that tumor necrosis factor (TNF) transitions astrocytes to an inflammatory reactive state that causes BBB dysfunction through activation of STAT3 and increased expression of SERPINA3, which encodes alpha 1-antichymotrypsin (α1ACT). To contextualize these findings, we correlated astrocytic STAT3 activation to vascular inflammation in postmortem human tissue. Further, in murine brain organotypic cultures, astrocyte-specific silencing of Serpina3n reduced vascular inflammation after TNF challenge. Last, treatment with recombinant Serpina3n in both ex vivo explant cultures and in vivo was sufficient to induce BBB dysfunction-related molecular changes. Overall, our results define the TNF-STAT3-α1ACT signaling axis as a driver of an inflammatory reactive astrocyte signature that contributes to BBB dysfunction.

CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states.

Astrocytes become reactive in response to insults to the central nervous system by adopting context-specific cellular signatures and outputs, but a systematic understanding of the underlying molecular mechanisms is lacking. In this study, we developed CRISPR interference screening in human induced pluripotent stem cell-derived astrocytes coupled to single-cell transcriptomics to systematically interrogate cytokine-induced inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures, one promoted by STAT3 and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in human brains in Alzheimer's disease and hypoxic-ischemic encephalopathy. Furthermore, we validated that markers of these signatures were regulated by STAT3 in vivo using a mouse model of neuroinflammation. These results and the platform that we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

Repurposing the cardiac glycoside digoxin to stimulate myelin regeneration in chemically-induced and immune-mediated mouse models of multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na+ /K+ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG35-55 (PLG-MOG35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.

Melanoma-Secreted Amyloid Beta Suppresses Neuroinflammation and Promotes Brain Metastasis.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared with those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (Aß) for growth and survival in the brain parenchyma. Melanoma-secreted Aß activates surrounding astrocytes to a prometastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacologic inhibition of Aß decreases brain metastatic burden. SIGNIFICANCE: Our results reveal a novel mechanistic connection between brain metastasis and Alzheimer's disease, two previously unrelated pathologies; establish Aß as a promising therapeutic target for brain metastasis; and demonstrate suppression of neuroinflammation as a critical feature of metastatic adaptation to the brain parenchyma. This article is highlighted in the In This Issue feature, p. 1171.

Monitoring phagocytic uptake of amyloid ß into glial cell lysosomes in real time.

Phagocytosis by glial cells is essential to regulate brain function during health and disease. Therapies for Alzheimer's disease (AD) have primarily focused on targeting antibodies to amyloid ß (Aß) or inhibitng enzymes that make it, and while removal of Aß by phagocytosis is protective early in AD it remains poorly understood. Impaired phagocytic function of glial cells during later stages of AD likely contributes to worsened disease outcome, but the underlying mechanisms of how this occurs remain unknown. We have developed a human Aß1-42 analogue (AßpH) that exhibits green fluorescence upon internalization into the acidic organelles of cells but is non-fluorescent at physiological pH. This allowed us to image, for the first time, glial uptake of AßpH in real time in live animals. We find that microglia phagocytose more AßpH than astrocytes in culture, in brain slices and in vivo. AßpH can be used to investigate the phagocytic mechanisms responsible for removing Aß from the extracellular space, and thus could become a useful tool to study Aß clearance at different stages of AD.

Neuroinflammatory astrocyte subtypes in the mouse brain.

Astrocytes undergo an inflammatory transition after infections, acute injuries and chronic neurodegenerative diseases. How this transition is affected by time and sex, its heterogeneity at the single-cell level and how sub-states are spatially distributed in the brain remains unclear. In this study, we investigated transcriptome changes of mouse cortical astrocytes after an acute inflammatory stimulus using the bacterial cell wall endotoxin lipopolysaccharide. We identified fast transcriptomic changes in astrocytes occurring within hours that drastically change over time. By sequencing ~80,000 astrocytes at single-cell resolution, we show that inflammation causes a widespread response with subtypes of astrocytes undergoing distinct inflammatory transitions with defined transcriptomic profiles. We also attribute key sub-states of inflammation-induced reactive astrocytes to specific brain regions using spatial transcriptomics and in situ hybridization. Together, our datasets provide a powerful resource for profiling astrocyte heterogeneity and will be useful for understanding the biological importance of regionally constrained reactive astrocyte sub-states.

Alterations in circulating extracellular vesicles underlie social stress-induced behaviors in mice.

Chronic stress induces peripheral and intracerebral immune changes and inflammation, contributing to neuropathology and behavioral abnormalities relevant to psychiatric disorders such as depression. Although the pathological implication of many peripheral factors such as pro-inflammatory cytokines, hormones, and macrophages has been demonstrated, the roles of circulating extracellular vesicles (EVs) for chronic stress mechanisms remain poorly investigated. Here, we report that chronic social defeat stress (CSDS)-induced social avoidance phenotype, assessed by a previously untested three-chamber social approach test, can be distinguished by multiple pro-inflammatory cytokines and EV-associated molecular signatures in the blood. We found that the expression patterns of miRNAs distinguished the CSDS-susceptible mice from the CSDS-resilient mice. Social avoidance behavior scores were also estimated with good accuracy by the expression patterns of multiple EV-associated miRNAs. We also demonstrated that EVs enriched from the CSDS-susceptible mouse sera upregulated the production of pro-inflammatory cytokines in the LPS-stimulated microglia-like cell lines. Our results indicate the role of circulating EVs and associated miRNAs in CSDS susceptibility, which may be related to pro-inflammatory mechanisms underlying stress-induced neurobehavioral outcomes.

CD49f Is a Novel Marker of Functional and Reactive Human iPSC-Derived Astrocytes.

New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f+ hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f+ hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f+ hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.

Complement 3+-astrocytes are highly abundant in prion diseases, but their abolishment led to an accelerated disease course and early dysregulation of microglia.

Astrogliosis and activation of microglia are hallmarks of prion diseases in humans and animals. Both were viewed to be rather independent events in disease pathophysiology, with proinflammatory microglia considered to be the potential neurotoxic species at late disease stages. Recent investigations have provided substantial evidence that a proinflammatory microglial cytokine cocktail containing TNF-α, IL-1α and C1qa reprograms a subset of astrocytes to change their expression profile and phenotype, thus becoming neurotoxic (designated as A1-astrocytes). Knockout or antibody blockage of the three cytokines abolish formation of A1-astrocytes, therefore, this pathway is of high therapeutic interest in neurodegenerative diseases. Since astrocyte polarization profiles have never been investigated in prion diseases, we performed several analyses and could show that C3+-PrPSc-reactive-astrocytes, which may represent a subtype of A1-astrocytes, are highly abundant in prion disease mouse models and human prion diseases. To investigate their impact on prion disease pathophysiology and to evaluate their potential therapeutic targeting, we infected TNF-α, IL-1α, and C1qa Triple-KO mice (TKO-mice), which do not transit astrocytes into A1, with prions. Although formation of C3+-astrocytes was significantly reduced in prion infected Triple-KO-mice, this did not affect the amount of PrPSc deposition or titers of infectious prions. Detailed characterization of the astrocyte activation signature in thalamus tissue showed that astrocytes in prion diseases are highly activated, showing a mixed phenotype that is distinct from other neurodegenerative diseases and were therefore termed C3+-PrPSc-reactive-astrocytes. Unexpectedly, Triple-KO led to a significant acceleration of prion disease course. While pan-astrocyte and -microglia marker upregulation was unchanged compared to WT-brains, microglial homeostatic markers were lost early in disease in TKO-mice, pointing towards important functions of different glia cell types in prion diseases.

Glutathione S-transferases promote proinflammatory astrocyte-microglia communication during brain inflammation.

Astrocytes and microglia play critical roles in brain inflammation. Here, we report that glutathione S-transferases (GSTs), particularly GSTM1, promote proinflammatory signaling in astrocytes and contribute to astrocyte-mediated microglia activation during brain inflammation. In vivo, astrocyte-specific knockdown of GSTM1 in the prefrontal cortex attenuated microglia activation in brain inflammation induced by systemic injection of lipopolysaccharides (LPS). Knocking down GSTM1 in astrocytes also attenuated LPS-induced production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by microglia when the two cell types were cocultured. In astrocytes, GSTM1 was required for the activation of nuclear factor κB (NF-κB) and the production of proinflammatory mediators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif chemokine ligand 2 (CCL2), both of which enhance microglia activation. Our study suggests that GSTs play a proinflammatory role in priming astrocytes and enhancing microglia activation in a microglia-astrocyte positive feedback loop during brain inflammation.

Extracellular Vesicles for Research on Psychiatric Disorders.

Extracellular vesicles (EVs) have gained increasing attention as underexplored intercellular communication mechanisms in basic science and as potential diagnostic tools in translational studies, particularly those related to cancers and neurological disorders. This article summarizes accumulated findings in the basic biology of EVs, EV research methodology, and the roles of EVs in brain cell function and dysfunction, as well as emerging EV studies in human brain disorders. Further research on EVs in neurobiology and psychiatry may open the door to a better understanding of intercellular communications in healthy and diseased brains, and the discovery of novel biomarkers and new therapeutic strategies in psychiatric disorders.

Behavioral Changes in Mice Lacking Interleukin-33.

Interleukin (IL)-33 is a member of the IL-1 family of cytokines. IL-33 is expressed in nuclei and secreted as alarmin upon cellular damage to deliver a danger signal to the surrounding cells. Previous studies showed that IL-33 is expressed in the brain and that it is involved in neuroinflammatory and neurodegenerative processes in both humans and rodents. Nevertheless, the role of IL-33 in physiological brain function and behavior remains unclear. Here, we have investigated the behaviors of mice lacking IL-33 (Il33-/- mice). IL-33 is constitutively expressed throughout the adult mouse brain, mainly in oligodendrocyte-lineage cells and astrocytes. Notably, Il33-/- mice exhibited reduced anxiety-like behaviors in the elevated plus maze (EPM) and the open field test (OFT), as well as deficits in social novelty recognition, despite their intact sociability, in the three-chamber social interaction test. The immunoreactivity of c-Fos proteins, an indicator of neuronal activity, was altered in several brain regions implicated in anxiety-related behaviors, such as the medial prefrontal cortex (mPFC), amygdala, and piriform cortex (PCX), in Il33-/- mice after the EPM. Altered c-Fos immunoreactivity in Il33-/- mice was not correlated with IL-33 expression in wild-type (WT) mice nor was IL-33 expression affected by the EPM in WT mice. Thus, our study has revealed that Il33-/- mice exhibit multiple behavioral deficits, such as reduced anxiety and impaired social recognition. Our findings also indicate that IL-33 may regulate the development and/or maturation of neuronal circuits, rather than control neuronal activities in adult brains.