Biology subjects

Cahalan, M. D.

Publications and source records attributed to Cahalan, M. D..

5 recordsLinked to original sources

bioRxiv2021

TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimers disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum (ER) stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.

neuroscience↗

bioRxiv2021

Impaired developmental microglial pruning of excitatory synapses on CRH-expressing hypothalamic neurons exacerbates stress responses throughout life

The developmental origins of stress-related mental illnesses are well-established, and early-life stress/adversity (ELA) is an important risk factor. However, it is unclear how ELA impacts the maturation of salient brain circuits, provoking enduring vulnerability to stress and stress-related disorders. Here we find that ELA increases the number and function of excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, and implicate disrupted synapse pruning by microglia as a key mechanism. Microglial process dynamics on live imaging, and engulfment of synaptic elements by microglia, were both attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor Mer. Accordingly, selective chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Selective early-life microglial activation also mitigated the adrenal hypertrophy and prolonged stress responses in adult ELA mice, establishing microglial actions during development as powerful contributors to experience-dependent sculpting of stress-related brain circuits.

neuroscience↗

bioRxiv2017

Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility

Ca2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration \"sparkles\" and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

immunology↗

bioRxiv2017

T Cell Calcium Dynamics Visualized in a Ratiometric tdTomato-GCaMP6f Transgenic Reporter Mouse

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here we describe development and characterization of \"Salsa6f\", a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f floxed and targeted to the Rosa26 locus for expression in specific cell types. Using CD4-Cre-Salsa6f mice, we report normal development and function of T cells expressing Salsa6f and demonstrate Ca2+ signaling dynamics during T cell receptor engagement in naive T cells, helper Th17 T cells and regulatory T cells. Salsa6f expression also revealed functional expression of mechanosensitive Piezo1 channels in T cells. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Deep tissue two-photon imaging of T cells in the steady-state lymph node revealed a highly localized Ca2+ signaling behavior (\"sparkles\") as cells migrate.

cell biology↗

bioRxiv2017

Cell-intrinsic activation of Orai1 regulates human T cell motility

Ca2+ signaling through the store-operated Ca2+ channel, Orai1, is crucial for T cell function, but a role in regulating T cell motility in lymph nodes has not been previously reported. Tracking human T cells in immunodeficient mouse lymph nodes and in microfabricated PDMS channels, we show that inhibition of Orai1 channel activity with a dominant-negative Orai1-E106A construct increases average T cell velocities by reducing the frequency of pauses in motile T cells. Orai1-dependent motility arrest occurs spontaneously during confined motility in vitro, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, we show these spontaneous pauses during T cell motility in vitro coincide with episodes of spontaneous cytosolic Ca2+ signaling. Our results demonstrate that Orai1, activated in a cell-intrinsic manner, regulates T cell motility patterns that accompany immune surveillance.

cell biology↗