The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons.

Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, we show that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.

Proteomic profiling of interferon-responsive reactive astrocytes in rodent and human.

Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNß and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.

Longitudinal scRNA-seq analysis in mouse and human informs optimization of rapid mouse astrocyte differentiation protocols.

Macroglia (astrocytes and oligodendrocytes) are required for normal development and function of the central nervous system, yet many questions remain about their emergence during the development of the brain and spinal cord. Here we used single-cell/single-nucleus RNA sequencing (scRNA-seq/snRNA-seq) to analyze over 298,000 cells and nuclei during macroglia differentiation from mouse embryonic and human-induced pluripotent stem cells. We computationally identify candidate genes involved in the fate specification of glia in both species and report heterogeneous expression of astrocyte surface markers across differentiating cells. We then used our transcriptomic data to optimize a previous mouse astrocyte differentiation protocol, decreasing the overall protocol length and complexity. Finally, we used multi-omic, dual single-nuclei (sn)RNA-seq/snATAC-seq analysis to uncover potential genomic regulatory sites mediating glial differentiation. These datasets will enable future optimization of glial differentiation protocols and provide insight into human glial differentiation.

Proteomic Alterations and Novel Markers of Neurotoxic Reactive Astrocytes in Human Induced Pluripotent Stem Cell Models.

Astrocytes respond to injury, infection, and inflammation in the central nervous system by acquiring reactive states in which they may become dysfunctional and contribute to disease pathology. A sub-state of reactive astrocytes induced by proinflammatory factors TNF, IL-1α, and C1q ("TIC") has been implicated in many neurodegenerative diseases as a source of neurotoxicity. Here, we used an established human induced pluripotent stem cell (hiPSC) model to investigate the surface marker profile and proteome of TIC-induced reactive astrocytes. We propose VCAM1, BST2, ICOSL, HLA-E, PD-L1, and PDPN as putative, novel markers of this reactive sub-state. We found that several of these markers colocalize with GFAP+ cells in post-mortem samples from people with Alzheimer's disease. Moreover, our whole-cells proteomic analysis of TIC-induced reactive astrocytes identified proteins and related pathways primarily linked to potential engagement with peripheral immune cells. Taken together, our findings will serve as new tools to purify reactive astrocyte subtypes and to further explore their involvement in immune responses associated with injury and disease.

Low FoxO expression in Drosophila somatosensory neurons protects dendrite growth under nutrient restriction.

During prolonged nutrient restriction, developing animals redistribute vital nutrients to favor brain growth at the expense of other organs. In Drosophila, such brain sparing relies on a glia-derived growth factor to sustain proliferation of neural stem cells. However, whether other aspects of neural development are also spared under nutrient restriction is unknown. Here we show that dynamically growing somatosensory neurons in the Drosophila peripheral nervous system exhibit organ sparing at the level of arbor growth: Under nutrient stress, sensory dendrites preferentially grow as compared to neighboring non-neural tissues, resulting in dendrite overgrowth. These neurons express lower levels of the stress sensor FoxO than neighboring epidermal cells, and hence exhibit no marked induction of autophagy and a milder suppression of Tor signaling under nutrient stress. Preferential dendrite growth allows for heightened animal responses to sensory stimuli, indicative of a potential survival advantage under environmental challenges.

The organs of a young animal develop in a carefully controlled way to reach the right size relative to each other. However, if the animal's diet does not contain the right amount of nutrients ­ a condition known as malnutrition ­ the body prioritizes the needs of the brain and other vital organs. This means that certain organs keep on growing while others stop. The brain is at the center of the nervous system, which is formed of networks of nerve cells (or neurons) that rapidly carry messages around the body. In the larvae of malnourished fruit flies, a molecular signal allows the nervous system to continue making new neurons as other parts of the body slow down their growth. During development, neurons also connect to each other by growing tree-like structures known as dendrites. However, it remained unclear whether the growth of dendrites was also protected during episodes of malnutrition. To address this question, Poe, Xu et al. performed experiments in the larvae of fruit flies, focusing on a type of neuron whose dendrites extend into the skin. When nutrients were scarce, the neurons grew more rapidly than the surrounding skin cells, resulting in dendrite overgrowth. Compared to neurons, the skin cells had higher levels of a stress sensor known as FoxO, which stops cell growth when nutrients are scarce. Conversely, low quantities of FoxO in neurons allow these cells to keep on growing dendrites, which ultimately helps the starved animals to better react to their environment. These results suggest that the growth of neurons and their connecting structures is preserved during malnutrition. Ultimately, dissecting how organisms prioritize resources can help to develop new approaches to treat human conditions that emerge during malnutrition.