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.

Project description:Despite effective antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are found in nearly one-third of patients. Using a cellular co-culture system including neurons and human microglia infected with HIV (hμglia/HIV), we investigated the hypothesis that HIV-dependent neurological degeneration results from the periodic emergence of HIV from latency within microglial cells in response to neuronal damage or inflammatory signals. When a clonal hμglia/HIV population (HC69) expressing HIV, or HIV infected human primary and iPSC-derived microglial cells, were cultured for a short-term (24 h) with healthy neurons, HIV was silenced. The neuron-dependent induction of latency in HC69 cells was recapitulated using induced pluripotent stem cell (iPSC)-derived GABAergic cortical (iCort) and dopaminergic (iDopaNer), but not motor (iMotorNer), neurons. By contrast, damaged neurons induce HIV expression in latently infected microglial cells. After 48-72 h co-culture, low levels of HIV expression appear to damage neurons, which further enhances HIV expression. There was a marked reduction in intact dendrites staining for microtubule associated protein 2 (MAP2) in the neurons exposed to HIV-expressing microglial cells, indicating extensive dendritic pruning. To model neurotoxicity induced by methamphetamine (METH), we treated cells with nM levels of METH and suboptimal levels of poly (I:C), a TLR3 agonist that mimics the effects of the circulating bacterial rRNA found in HIV infected patients. This combination of agents potently induced HIV expression, with the METH effect mediated by the σ1 receptor (σ1R). In co-cultures of HC69 cells with iCort neurons, the combination of METH and poly(I:C) induced HIV expression and dendritic damage beyond levels seen using either agent alone, Thus, our results demonstrate that the cross-talk between healthy neurons and microglia modulates HIV expression, while HIV expression impairs this intrinsic molecular mechanism resulting in the excessive and uncontrolled stimulation of microglia-mediated neurotoxicity.

Dataset Information

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Publications

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

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