Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state, microglia nucleate microtubules at Golgi outposts, and activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease

Microglia control glutamatergic synapses in the adult mouse hippocampus

Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1−/− mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses

Microglia reactivity entails microtubule remodeling from acentrosomal to centrosomal arrays

Microglia are the resident immune cells of the brain, crucial for the maintenance of the homeostasis in the central nervous system. They are responsible for removing cellular debris, pathogens, and damaged cells, as well as modulating neuronal function. Microglia actively and continuously patrol their local environment by using highly motile processes and ramified morphology. In response to triggering stimuli, such as infection, altered neuronal activity, trauma or neurodegenerative diseases, microglia undergo a process of activation that profoundly change their gene expression and functions. Microglia activation is a salient feature of neuroinflammation, prominent in many neurodegenerative diseases. Interestingly, microglia reactivity also entails a large-scale remodeling of cellular geometry, but while the role of the actin cytoskeleton in driving these morphological changes and the specialized functions of activated microglia has been extensively studied, the behavior of the microtubule cytoskeleton during these changes remains unexplored. Microtubules are hollow, cylindrical polymers composed of protofilaments of α/β tubulin heterodimers that arrange in a head-to-tail fashion, making microtubule polarized structures with a fast growing plus end and a slow growing minus end. Microtubules form the backbone of the cell's internal transport system for macromolecules and organelles and are also involved in cell division and the maintenance of cell shape and integrity. We hypothesize that rearrangement of the microtubule cytoskeleton would play a major role in the morphological changes guiding microglia transition from homeostatic to activated states providing a novel target to modulate microglia activation. Through a detailed in vitro phenotyping approach and validation in mouse retinal tissue we report that: 1) in homeostatic conditions, ramified microglia possess stable microtubule arrays, while microglia activation increases microtubule dynamic behavior. 2) A non-centrosomal microtubule organization in arrays with mixed polarity is a hallmark of homeostatic microglia, similar to the architecture typical of highly specialized cells such as neurons and oligodendrocytes, while activation induces recruitment of the majority of microtubule minus end at the centrosome. 3) Homeostatic microglia nucleate non-centrosomal microtubules at Golgi outposts, similar to what occurs in neurons, while activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results unveil the remodeling of the microtubule cytoskeleton as a striking hallmark of microglia reactivity and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of activated microglia, inhibition of microtubule dynamics may provide a novel strategy to reduce microglia reactivity in inflammatory disease

Biphenyl-3-yl alkylcarbamates as fatty acid amide hydrolase (FAAH) inhibitors: steric effects of N-alkyl chain on rat plasma and liver stability

Secondary alkylcarbamic acid biphenyl-3-yl esters are a class of Fatty Acid Amide Hydrolase (FAAH) inhibitors, which include the reference compounds URB597 and URB694. Given the intrinsic reactivity of the carbamate group, the in vivo potency of these molecules in rats is strongly affected by their hydrolysis in plasma or hepatic metabolism. In the present study, in vitro chemical and metabolic stability assays (rat plasma and rat liver S(9) fraction) were used to investigate the structure-property relationships (SPRs) for a focused series of title compounds, where lipophilicity and steric hindrance of the carbamate N-substituent had been modulated. The resulting degradation rates indicate that a secondary or tertiary alkyl group at the carbamate nitrogen atom increases hydrolytic stability towards rat plasma esterases. The calculated solvent accessible surface area (SASA) of the carbamate fragment was employed to describe the differences observed in rate constants of hydrolysis in rat plasma (log k(plasma)), suggesting that stability in plasma increases if the substituent exerts a shielding effect on the carbamate carbonyl. Stability in rat liver S(9) fraction is increased when a tertiary carbon is bound to the carbamate nitrogen atom, while other steric effects showed complex relationships with degradation rates. The SPRs here described may be applied at the pharmacokinetic optimization of other classes of carbamate FAAH inhibitors

Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells

The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial-mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells' infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment

Diagnosis of West Nile Virus Human Infections: Overview and Proposal of Diagnostic Protocols Considering the Results of External Quality Assessment Studies

West Nile virus, genus Flavivirus, is transmitted between birds and occasionally other animals by ornithophilic mosquitoes. This virus also infects humans causing asymptomatic infections in about 85% of cases and <1% of clinical cases progress to severe neuroinvasive disease. The virus also presents a threat since most infections remain unapparent. However, the virus contained in blood and organs from asymptomatically infected donors can be transmitted to recipients of these infectious tissues. This paper reviews the presently available methods to achieve the laboratory diagnosis of West Nile virus infections in humans, discussing the most prominent advantages and disadvantages of each in light of the results obtained during four different External Quality Assessment studies carried out by the European Network for ‘Imported’ Viral Diseases (ENIV

Antibiotics Treatment Modulates Microglia–Synapses Interaction

‘Dysbiosis’ of the adult gut microbiota, in response to challenges such as infection, altered diet, stress, and antibiotics treatment has been recently linked to pathological alteration of brain function and behavior. Moreover, gut microbiota composition constantly controls microglia maturation, as revealed by morphological observations and gene expression analysis. However, it is unclear whether microglia functional properties and crosstalk with neurons, known to shape and modulate synaptic development and function, are influenced by the gut microbiota. Here, we investigated how antibiotic-mediated alteration of the gut microbiota influences microglial and neuronal functions in adult mice hippocampus. Hippocampal microglia from adult mice treated with oral antibiotics exhibited increased microglia density, altered basal patrolling activity, and impaired process rearrangement in response to damage. Patch clamp recordings at CA3-CA1 synapses revealed that antibiotics treatment alters neuronal functions, reducing spontaneous postsynaptic glutamatergic currents and decreasing synaptic connectivity, without reducing dendritic spines density. Antibiotics treatment was unable to modulate synaptic function in CX3CR1-deficient mice, pointing to an involvement of microglia–neuron crosstalk through the CX3CL1/CX3CR1 axis in the effect of dysbiosis on neuronal functions. Together, our findings show that antibiotic alteration of gut microbiota impairs synaptic efficacy, suggesting that CX3CL1/CX3CR1 signaling supporting microglia is a major player in in the gut–brain axis, and in particular in the gut microbiota-to-neuron communication pathway