Neuroinflammation plays a critical role in the onset and progression of many neurological disorders, including Multiple Sclerosis, Alzheimer's and Parkinson's diseases. In these clinical conditions the underlying neuroinflammatory processes are significantly heterogeneous. Nevertheless, a common link is the chronic activation of innate immune responses and imbalanced secretion of pro and anti-inflammatory mediators. In light of this, the discovery of robust biomarkers is crucial for screening, early diagnosis, and monitoring of neurological diseases. However, the difficulty to investigate biochemical processes directly in the central nervous system (CNS) is challenging. In recent years, biomarkers of CNS inflammatory responses have been identified in different body fluids, such as blood, cerebrospinal fluid, and tears. In addition, progress in micro and nanotechnology has enabled the development of biosensing platforms capable of detecting in real-time, multiple biomarkers in clinically relevant samples. Biosensing technologies are approaching maturity where they will become deployed in community settings, at which point screening programs and personalized medicine will become a reality. In this multidisciplinary review, our goal is to highlight both clinical and recent technological advances toward the development of multiplex-based solutions for effective neuroinflammatory and neurodegenerative disease diagnostics and monitoring.

Summary This protocol highlights the use of FRET-based biosensors to investigate signaling events during microglia activation in real time. Understanding microglia activation has gained momentum as it can help decipher signaling mechanisms underlying the neurodegenerative process occurring in neurological disorders. Unlike more traditional methods widely employed in the microglia field, FRET allows microglia signaling events to be studied in real time with exquisite subcellular resolution. However, FRET-based live-cell imaging requires application-specific biosensors and specialized imaging systems, limiting its use in in vivo studies. For complete details on the use and execution of this protocol, please refer to Socodato et al. (2020) , Portugal et al. (2017) , and Socodato et al. (2018) .

The ramified morphology of microglia and the dynamics of their membrane protrusions are essential for their functions in central nervous system development, homeostasis, and disease. Although their ability to change and control shape critically depends on the actin and actomyosin cytoskeleton, the underlying regulatory mechanisms remain largely unknown. In this study, we systematically analyzed the actomyosin cytoskeleton and regulators downstream of the small GTPase RhoA in the control of microglia shape and function. Our results reveal that (i) Myh9 controls cortical tension levels and affects microglia protrusion formation, (ii) cofilin-mediated maintenance of actin turnover regulates microglia protrusion extension, and (iii) Myh10 influences microglia inflammatory activation. Overall we uncover molecular pathways that regulate microglia morphology and identify type-II myosins as important regulators of microglia biology with differential roles in the control of cell shape (Myh9) and functions (Myh10).

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