Periodic Reporting for period 2 - iMIND (iPS-derived MIcroglia and Neuroinflammation in Dementia)
Período documentado: 2022-02-20 hasta 2023-02-19
The iMIND project is structured to investigate the contribution of neuroinflammation in Alzheimer’s disease (AD) by employing a novel, human-derived, pathology-relevant cellular model. AD is an irreversible neurodegenerative condition affecting 50 million people worldwide. The current figure indicates that AD is becoming a severe threat to the health system's stability. However, no disease-modifying strategies are available despite major research and clinical efforts. Neuroinflammation is emerging as a key component of the disease, affecting its onset and progression. Recent analyses have identified over 20 genes that influence the risk of developing AD and, of note, most of these genes are primarily expressed in microglia, the innate immune cells of the brain and critical modulators of neuroinflammatory processes.
The original proposal was set to investigate a rare protective mutation (P522A) in the PLCG2 (Phospholipase C Gamma 2) gene associated with AD in a model of microglia derived from human pluripotent stem cells (iPSC). Unfortunately, the early phases of the fellowship have been significantly impacted by the COVID-19 pandemic and the stringent plans set in place to limit its spread. Therefore, a contingency plan was drafted to limit the disruption of research activities. The revised proposal focused on the investigation and the functional characterization of the TREM2 gene in human microglia. Like PLCG2, the TREM2 gene is primarily expressed in microglia but, unlike PLCG2, rare loss-of-function TREM2 variants increase the risk of developing AD. We have previously shown that TREM2 knock-out (TREM2 KO) alters gene expression and functional responses in human AD models. However, the molecular mechanisms linking deficits in TREM2 signaling to the functional alterations observed in AD are still poorly understood. The key objectives of the iMIND project are as follow: 1) to gain expertise in the generation of microglia derived from wild-type and TREM2 KO mutant iPSC, 2) to investigate the molecular and functional responses of mutant microglia to AD-related stimuli, 3) to investigate functional effects of TREM2 deficits on calcium (Ca2+) signaling and cell motility. Additional objectives, specifically investigated during the return phase of the fellowship, are as follow: 1) to detect the specific source of Ca2+ that contributes to the spontaneous transients and 2) to investigate the contribution of Ca2+ signaling on directed cell migration in human microglia.
Collectively, the project identified novel therapeutic targets, explored new grounds in the molecular underpinnings of AD, and elucidated key, unexpected mechanisms of microglia functioning in physiological and pathological settings.
The original proposal was set to investigate a rare protective mutation (P522A) in the PLCG2 (Phospholipase C Gamma 2) gene associated with AD in a model of microglia derived from human pluripotent stem cells (iPSC). Unfortunately, the early phases of the fellowship have been significantly impacted by the COVID-19 pandemic and the stringent plans set in place to limit its spread. Therefore, a contingency plan was drafted to limit the disruption of research activities. The revised proposal focused on the investigation and the functional characterization of the TREM2 gene in human microglia. Like PLCG2, the TREM2 gene is primarily expressed in microglia but, unlike PLCG2, rare loss-of-function TREM2 variants increase the risk of developing AD. We have previously shown that TREM2 knock-out (TREM2 KO) alters gene expression and functional responses in human AD models. However, the molecular mechanisms linking deficits in TREM2 signaling to the functional alterations observed in AD are still poorly understood. The key objectives of the iMIND project are as follow: 1) to gain expertise in the generation of microglia derived from wild-type and TREM2 KO mutant iPSC, 2) to investigate the molecular and functional responses of mutant microglia to AD-related stimuli, 3) to investigate functional effects of TREM2 deficits on calcium (Ca2+) signaling and cell motility. Additional objectives, specifically investigated during the return phase of the fellowship, are as follow: 1) to detect the specific source of Ca2+ that contributes to the spontaneous transients and 2) to investigate the contribution of Ca2+ signaling on directed cell migration in human microglia.
Collectively, the project identified novel therapeutic targets, explored new grounds in the molecular underpinnings of AD, and elucidated key, unexpected mechanisms of microglia functioning in physiological and pathological settings.
To achieve the key objectives of the project we employed iPSC lines gene edited via CRISPR/Cas9 technology to knock-out the TREM2 gene. After validation, generated isogenic iPSC lines have been differentiated into induced microglia (hereafter termed iMGL) as described in McQuade et al. (2018, Mol Neurodegen). Compelling evidence indicates that defective TREM2 signaling impairs microglia ability to sense and detect noxious stimuli resulting in an impaired cellular response in AD pathology. Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in iMGL. Changes in spontaneous and evoked intracellular Ca2+ levels were monitored in WT and TREM2 KO loaded with selective, high-affinity Ca2+ indicators. Overall, our findings suggest that TREM2 KO iMGL show exaggerated Ca2+ signals in response to cell damage-related molecules, like purinergic signaling agonists (i.e. ADP, ATP, UDP), instrumental for shaping microglia response to injury. This hypersensitivity to purinergic signaling is driven by an increased expression of P2Y12 and P2Y13 receptors, which in turn results in a greater release of Ca2+ from the endoplasmic reticulum (ER) and a subsequent, sustained Ca2+ influx through Orai1 channels. To couple these Ca2+ signaling alterations with the phenotypic traits observed in TREM2 KO, we developed a new cell line expressing the genetically encoded Ca2+ probe Salsa6f and found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Motility analyses indicate that TREM2 KO iMGL displayed greater overall displacement but showed impaired chemotaxis upon ADP stimulation. This chemotactic defect can be rescued by reducing cytosolic Ca2+ rises by employing ADP receptor antagonists. The study also prompted the implementation of a set of new analytical tools for the assessment and quantification of cell migration/motility.
The present results have been included in a recent publication in the international peer-reviewed journal eLife (Jairaman, McQuade et al., 2022). An additional original paper is in preparation and investigated the basic principles of directed cell migration in iMGL. The results have been the object of presentations at international conferences and local/national/international seminars. Results achieved during the development of the fellowship have been also presented during the activities of the European Researcher’s Night organized at the host institution.
The present results have been included in a recent publication in the international peer-reviewed journal eLife (Jairaman, McQuade et al., 2022). An additional original paper is in preparation and investigated the basic principles of directed cell migration in iMGL. The results have been the object of presentations at international conferences and local/national/international seminars. Results achieved during the development of the fellowship have been also presented during the activities of the European Researcher’s Night organized at the host institution.
Our findings demonstrate that loss of TREM2 contributes to a dysregulation of Ca2+ homeostasis in microglia in response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility. The study, therefore, prompted the identification of novel molecular pathways that might be involved in AD development and that could represent promising therapeutic targets aimed at modulating microglia response in disease-relevant settings. The project also has a technological impact as we developed a new iPSC-derived iMGL cell line expressing the genetically encoded Ca2+ probe Salsa6f, a powerful tool set to investigate the role of microglia Ca2+ signaling as well as cellular dynamics in health and disease.