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Microarray analysis to evaluate the role of USP18 in primary microglia and the microglia cell line BV-2

ABSTRACT: Microglia are tissue macrophages of the central nervous system (CNS) that control tissue homeostasis, and as such they are crucially important for organ integrity. Microglia dysregulation is thought to be causal for a group of neuropsychiatric, neurodegenerative and neuroinflammatory diseases, called ‘microgliopathies’. However, how the intracellular stimulation machinery in microglia is controlled is poorly understood. By using expression studies, we identified the ubiquitin-specific protease (Usp) 18 in white matter microglia that essentially contributes to microglial quiescence under homeostatic conditions. We further found that microglial Usp18 negatively regulated the activation of STAT1 and concomitant induction of interferon-induced genes thereby disabling the termination of IFN signalling. Unexpectedly, the Usp18-mediated feedback loop was independent from the catalytic domain of the protease but instead required the interacting region of Ifnar2. Additionally, the absence of Ifnar1 completely rescued microglial activation indicating a tonic IFN signal mediated by receptor interactions under non-diseased conditions. Finally, conditional depletion of Usp18 only in myeloid cells significantly enhanced the disease burden in a mouse model of CNS autoimmunity, increased axonal and myelin damage and determined the spatial distributions of CNS lesions that shared the same STAT1 signature as myeloid cells found in active multiple sclerosis (MS) lesions. These results identify Usp18 as novel negative regulator of microglia activation, demonstrate a protective role of the IFNAR pathway for microglia and establish Usp18 as potential therapeutic target for the treatment of MS.

ORGANISM(S): Mus musculus

PROVIDER: GSE61500 | GEO | 2015/04/20

SECONDARY ACCESSION(S): PRJNA261321

REPOSITORIES: GEO

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Project description:Multiple Sclerosis (MS) is a leading cause of incurable progressive disability in young adults caused by inflammation in the central nervous system (CNS) that triggers demyelination, glial cell dysfunction and irreversible neuro-axonal damage1. While considerable progress has been made in treating early inflammatory relapsing-remitting MS, the mechanisms underpinning the progressive stage remain largely unknown. The capacity of microglia, the CNS-resident phagocytes, to clear tissue debris is essential for both maintaining and restoring CNS homeostasis2,3 and this capacity diminishes with age4-6. Age strongly associates with the risk of developing progressive MS7,8. Herein we demonstrate that the recovery from inflammation is dependent on the ability of microglia to clear tissue debris and that blocking this process leads to development of progressive disease in a murine model of MS. Microglia-specific deletion of the general autophagy regulator Atg7, but not the canonical macroautophagy protein Ulk1, led to increased intracellular accumulation of phagocytosed myelin. This further associated with the alteration of the microglial phenotype towards that previously described in other neurodegenerative diseases2,3,9. Moreover, Atg7 deficient microglia showed striking similarities with microglia from aged wild type mice, which also demonstrated accumulation of myelin debris and inability to recover from MS-like disease. In contrast, the induction of autophagy using the disaccharide Trehalose in aged mice led to functional myelin clearance and remission from MS-like disease. Our results demonstrate that a non-canonical form of autophagy in microglia is responsible for myelin clearance and that impairment of this pathway markedly changes microglial phenotype and prevents recovery from MS-like disease. Importantly, we show that using a disaccharide ubiquitously found in plant-derived foods to boost autophagy in cells in which this process is naturally diminished can be utilized for therapeutic purposes.Project was run on 4 lanes with a 10M sequencing depth

Project description:SPEACC-seq is a novel high-throughput method which enables forward genetic screens to identify cell-cell interaction mechanisms that uncovered an astrocyte-microglia regulatory circuit mediated by amphiregulin and IL33-ST2. signaling. Cell-cell interactions in the central nervous system (CNS) play central roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. For example, several factors mediate microglia-astrocyte interactions that promote CNS pathology, but less is known about regulatory interactions that limit tissue pathology. Here we report the development of SPEACC-seq (Stimulation, Perturbation, and Encapsulation of interACting Cells followed by Sequencing), a forward genetic screening platform which combines genome-wide CRISPR/Cas9 perturbations, cell co-culture in picoliter droplets, and microfluidic-based fluorescence activated droplet sorting to identify mechanisms of cell-cell communication. Using SPEACC-seq in combination with an in vivo perturb-seq screen, we identified microglia-produced amphiregulin as a suppressor of disease promoting astrocyte responses in experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis (MS). The production of microglial amphiregulin was induced via ST2 signaling by IL-33 released from astrocytes during EAE. Indeed, the genetic inactivation of ST2 or amphiregulin in microglia, or IL-33 or amphiregulin signaling in astrocytes resulted in the worsening of EAE, suggesting that IL-33-induced microglial amphiregulin limits disease-promoting astrocyte responses associated with CNS pathology. This regulatory loop was also detected in human astrocytes and microglia both in vitro and in MS patient CNS samples. In summary, we developed SPEACC-seq, a high-throughput, droplet-based forward genetic screening platform for the identification of cell-cell interaction mechanisms, which identified a novel microglia-astrocyte negative feedback loop that limits CNS pathology.

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Microarray analysis to evaluate the role of USP18 in primary microglia and the microglia cell line BV-2

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