Ambrosio, A. F. | Search bioRxiv

bioRxiv2025

NDR2 Kinase Regulate Microglial Metabolic Adaptation and Inflammatory Response: Critical Role in Glucose-Dependent Functional Plasticity

Diabetes and its retinal complication, diabetic retinopathy (DR), are a rapidly increasing health, societal and economic burden. Diabetic retinopathy is a complex disease with a chronic inflammatory component mediated by retinal microglial cells. Recent studies have demonstrated the importance of the Hippo pathway kinases, Ndr1/Stk38 and Ndr2/Stk38l, in the regulation of macrophages, immune cells that share similarities with microglial cells. However, the role of NDR2 kinases in microglial inflammatory response and in the pathophysiology of diabetic retinopathy has not yet been uncovered. This study investigates the role of NDR2 kinase in microglial cells, particularly in response to high glucose (HG) conditions. Using CRISPR-Cas9, we downregulated Ndr2 kinase in BV-2 microglial cells and analyzed the impact on cellular metabolism, phagocytosis and migratory capabilities. We demonstrate that microglial cells expressed NDR2 kinase protein, especially in HG conditions, suggesting its importance in regulating microglial functions during hyperglycemia. Ndr2 downregulated cells present a decreased basal respiration, indicating an impaired mitochondrial function. They also showed decreased metabolic flexibility to stress conditions, such as adaptation to HG conditions. Functionally, Ndr2 downregulation led to decreased phagocytic capacity and migration of microglial cells, both cytoskeleton-based functions. Furthermore, Ndr2 downregulation resulted in altered cytokine and chemokine secretion profiles. Notably, increased levels of pro-inflammatory cytokines such as IL-6, TNF, IL-17 and IL-12p70 were observed in Ndr2 downregulated cells, even under normal glucose conditions. In conclusion, our findings indicate that NDR2 kinase is crucial for microglial metabolic adaptation to stress, such as high glucose exposure and for influencing microglial inflammatory responses. Therefore, NDR2 kinase plays a vital role in maintaining microglial functional plasticity in response to glucose variations, suggesting potential implications for neuroinflammatory processes in conditions like diabetic retinopathy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/659313v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1380275org.highwire.dtl.DTLVardef@2950f9org.highwire.dtl.DTLVardef@833593org.highwire.dtl.DTLVardef@325171_HPS_FORMAT_FIGEXP M_FIG C_FIG Research in Context What is already known about this subject?O_LIThe pathogenesis of diabetic retinopathy (DR) involves chronic inflammation mediated by retinal microglial cells, which contribute to vascular damage and neurodegeneration. C_LIO_LIMicroglial dysfunction under high glucose (HG) conditions exacerbates cytokine release and oxidative stress, driving DR progression. C_LIO_LINDR kinases regulate inflammatory pathways in macrophages, but their role in microglia during DR was previously unexplored. C_LI What is the key question?O_LIHow do NDR2 kinase regulate microglial inflammatory responses and functional adaptability in diabetic retinopathy? C_LI What are the new findings?O_LINDR2 expression is upregulated in microglia exposed to HG. C_LIO_LINdr2 downregulation in microglia impairs metabolic flexibility, phagocytosis, and migration. C_LIO_LINdr2 downregulation disrupts cytoskeleton-dependent microglial functions, limiting their ability to adapt to metabolic stress. C_LIO_LINdr2 downregulation in microglia increases pro-inflammatory cytokines (IL-17, TNF) and reduces anti-inflammatory factors (sTNFRI, VEGF), exacerbating inflammation. C_LI How might this impact clinical practice?O_LITargeting Ndr2 signaling could emerge as a therapeutic strategy to modulate microglial-driven inflammation, potentially slowing DR progression and complementing existing glycemic control approaches. C_LI

immunology↗

bioRxiv2021

Microglia remodel the synaptic signaling required for context-dependent cognitive performance

Microglial homeostatic functions are fundamental to regulate the central nervous system microenvironment. We use conditional cell-specific gene targeting, RNA-seq profiling, high-throughput proteomics, phosphoproteomics, systems biology, and animal behavior to report a critical role for the RhoGTPase Rac1 in regulating adult microglia physiology. Ablation of Rac1 in adult microglia impaired their ability to sense and interpret the brain microenvironment and affected their capacity to communicate with synapses to drive cognitive performance, both at the steady-state and during experience-dependent plasticity. Overall, our results reveal a novel and central role for Rac1 as a regulator of microglia homeostasis and a molecular driver of the microglia-synapse crosstalk required for context-dependent sociability and learning related to memory.

neuroscience↗

bioRxiv2021

MorphData: Automating the data extraction process of morphological features of microglial cells in ImageJ

Microglial cells are the first line of defense within the central nervous system, with morphological characterization being widely used to define their activation status. Most methods to evaluate microglia status are manual, and, therefore, often biased, inaccurate, and time consuming. In fact, the process to collect morphological data starts with the acquisition of photomicrographs from where images of single cells are extracted. Then, the researcher collects the morphological features that characterize each cell. However, a manual data collection process from single cells can take weeks to complete. This work describes an open-source ImageJ plugin, MorphData, which automatizes the data extraction process of morphological features of single microglial cells. The plugin collects, processes, and organizes features associated with cell complexity and ramification. In a computer with limited computing power, MorphData was able to handle 699 single cells in less than 14 minutes. The same process, if performed manually, would take almost 19 working days. Overall, MorphData significantly reduces the time taken to collect morphological data from microglial cells, which can then be used to study, understand, and characterize microglia behavior in the brain of human patients or of animal models of neurological and psychiatric diseases.

bioinformatics↗

bioRxiv2017

Genetic ablation of RhoA in adult microglia causes synapse and neuronal loss

Nervous tissue homeostasis requires regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation producing a neurological phenotype (including synapse and neuron loss, impairment of LTP, formation of {beta}-amyloid plaques and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated Tnf production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also found that the Rhoa/Src signaling pathway was disrupted in microglia of the APP/PS1 mouse model of Alzheimers disease and that low doses of A{beta} oligomers triggered microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing RhoGTPase signaling in microglia can directly cause neurodegeneration.

neuroscience↗