BV2-derived extracellular vesicles modulate microglia inflammatory profile, neuronal plasticity, and behavioural performances in late adult mice.

BACKGROUND: During aging, both the brain and the immune system undergo a progressive impairment of physiological functions. Microglia, the immunocompetent cells of the central nervous system, shift towards a chronic mild inflammatory state that impacts brain homeostasis. Extracellular vesicles (EVs) released by microglia transport packages of molecular information that mirror the inflammatory status of donor cells and modulate the inflammatory phenotype of recipient microglia and other cell types. RESULTS: We demonstrated that intranasal administration of EVs derived from microglial-like BV2 cells to late adult mice (16-20 months of age) shifts microglia toward a "juvenile" morphology affecting their inflammatory profile. Mice treated with BV2-derived EVs have a reduction of anxiety-like behavior and an increased spatial learning, with sex-dependent differences. Further, BV2-derived EVs increased neuronal plasticity both in male and female mice. These findings suggest the involvement of microglial cells in vesicles-mediated anti-aging effect. CONCLUSIONS: Our data indicate that BV2-derived EVs could represent a resource to slow down age-dependent inflammation in the mouse brain.

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.

Optonongenetic enhancement of activity in primary cortical neurons.

It has been recently demonstrated that the exposure of naive neuronal cells to light-at the basis of optogenetic techniques and calcium imaging measurements-may alter neuronal firing. Indeed, understanding the effect of light on nongenetically modified neurons is crucial for a correct interpretation of calcium imaging and optogenetic experiments. Here we investigated the effect of continuous visible LED light exposure (490 nm, $ 0.18 {-} 1.3\;{\rm mW}/{{\rm mm}^2} $0.18-1.3mW/mm2) on spontaneous activity of primary neuronal networks derived from the early postnatal mouse cortex. We demonstrated, by calcium imaging and patch clamp experiments, that illumination higher than $ 1.0\;{\rm mW}/{{\rm mm}^2} $1.0mW/mm2 causes an enhancement of network activity in cortical cultures. We investigated the possible origin of the phenomena by blocking the transient receptor potential vanilloid 4 (TRPV4) channel, demonstrating a complex connection between this temperature-dependent channel and the measured effect. The results presented here shed light on an exogenous artifact, potentially present in all calcium imaging experiments, that should be taken into account in the analysis of fluorescence data.

Current multiple sclerosis treatments have improved our understanding of MS autoimmune pathogenesis.

Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults. When MS is not treated, it leads to irreversible and severe disability. The etiology of MS and its pathogenesis are not fully understood. The recent discovery that MS-associated genetic variants code for molecules related to the function of specific immune cell subsets is consistent with the concept of MS as a prototypic, T-cell-mediated autoimmune disease targeting the CNS. While the therapeutic efficacy of the currently available immunomodulatory therapies further strengthen this concept, differences observed in responses to MS treatment as well as additional clinical and imaging observations have also shown that the autoimmune pathogenesis underlying MS is much more complex than previously thought. There is therefore an unmet need for continued detailed phenotypic and functional analysis of disease-relevant adaptive immune cells and tissues directly derived from MS patients to unravel the immune etiology of MS in its entire complexity. In this review, we will discuss the currently available MS treatment options and approved drugs, including how they have contributed to the understanding of the immune pathology of this autoimmune disease.

Antibiotics treatment promotes vasculogenesis in the brain of glioma-bearing mice.

In recent years, several studies described the close relationship between the composition of gut microbiota and brain functions, highlighting the importance of gut-derived metabolites in mediating neuronal and glial cells cross-talk in physiological and pathological condition. Gut dysbiosis may affects cerebral tumors growth and progression, but the specific metabolites involved in this modulation have not been identified yet. Using a syngeneic mouse model of glioma, we have investigated the role of dysbiosis induced by the administration of non-absorbable antibiotics on mouse metabolome and on tumor microenvironment. We report that antibiotics treatment induced: (1) alteration of the gut and brain metabolome profiles; (2) modeling of tumor microenvironment toward a pro-angiogenic phenotype in which microglia and glioma cells are actively involved; (3) increased glioma stemness; (4) trans-differentiation of glioma cells into endothelial precursor cells, thus increasing vasculogenesis. We propose glycine as a metabolite that, in ABX-induced dysbiosis, shapes brain microenvironment and contributes to glioma growth and progression.

CXCL16 orchestrates adenosine A3 receptor and MCP-1/CCL2 activity to protect neurons from excitotoxic cell death in the CNS.

A role for chemokines as molecules mediating neuron-glia cross talk has emerged in recent years, both in physiological and pathological conditions. We demonstrate here for the first time that the chemokine CXCL16 and its unique receptor CXCR6 are functionally expressed in the CNS, and induce neuroprotection against excitotoxic damage due to excessive glutamate (Glu) exposure and oxygen glucose deprivation (OGD). In mice and rats we found that, to exert neuroprotection, CXCL16 requires the presence of extracellular adenosine (ADO), and that pharmacological or genetic inactivation of the ADO A(3) receptor, A(3)R, prevents CXCL16 effect. In experiments with astrocytes cocultured with cxcr6(gfp/gfp) hippocampal cells, we demonstrate that CXCL16 acts directly on astrocytes to release soluble factors that are essential to mediate neuroprotection. In particular, we report that (1) upon stimulation with CXCL16 astrocytes release monocyte chemoattractant protein-1/CCL2 and (2) the neuroprotective effect of CXCL16 is reduced in the presence of neutralizing CCL2 antibody. In conclusion, we found that chemokine CXCL16 is able to mediate cross talk between astrocytes and neighboring neurons and, in pathological conditions such as excessive Glu or OGD exposure, is able to counteract neuronal cell death through an ADO-dependent chemokine-induced chemokine-release mechanism.

Protocol for observing microtubules and microtubule ends in both fixed and live primary microglia cells.

Microtubule dynamics and orientation have crucial roles in many vital cellular processes. However, functional live imaging of microtubules and/or microtubule ends in primary microglia can be challenging. Here, we present a protocol for observing microtubules and microtubule ends in both fixed and live primary microglia cells. We describe steps for microglia culture and in vitro stimulation, SiR-tubulin labeling, lentivirus preparation, live imaging, immunostaining, and image acquisition. We also provide procedures for SiR-tubulin, EB3-EGFP, and EB1 analyses. For complete details on the use and execution of this protocol, please refer to Rosito et al. (2023).1.

Collective behavior and self-organization in neural rosette morphogenesis.

Neural rosettes develop from the self-organization of differentiating human pluripotent stem cells. This process mimics the emergence of the embryonic central nervous system primordium, i.e., the neural tube, whose formation is under close investigation as errors during such process result in severe diseases like spina bifida and anencephaly. While neural tube formation is recognized as an example of self-organization, we still do not understand the fundamental mechanisms guiding the process. Here, we discuss the different theoretical frameworks that have been proposed to explain self-organization in morphogenesis. We show that an explanation based exclusively on stem cell differentiation cannot describe the emergence of spatial organization, and an explanation based on patterning models cannot explain how different groups of cells can collectively migrate and produce the mechanical transformations required to generate the neural tube. We conclude that neural rosette development is a relevant experimental 2D in-vitro model of morphogenesis because it is a multi-scale self-organization process that involves both cell differentiation and tissue development. Ultimately, to understand rosette formation, we first need to fully understand the complex interplay between growth, migration, cytoarchitecture organization, and cell type evolution.

The choroid plexus acts as an immune cell reservoir and brain entry site in experimental autoimmune encephalomyelitis.

The choroid plexus (ChP) has been suggested as an alternative central nervous system (CNS) entry site for CCR6+ Th17 cells during the initiation of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). To advance our understanding of the importance of the ChP in orchestrating CNS immune cell entry during neuroinflammation, we here directly compared the accumulation of CD45+ immune cell subsets in the ChP, the brain and spinal cord at different stages of EAE by flow cytometry. We found that the ChP harbors high numbers of CD45int resident innate but also of CD45hi adaptive immune cell subsets including CCR6+ Th17 cells. With the exception to tissue-resident myeloid cells and B cells, numbers of CD45+ immune cells and specifically of CD4+ T cells increased in the ChP prior to EAE onset and remained elevated while declining in brain and spinal cord during chronic disease. Increased numbers of ChP immune cells preceded their increase in the cerebrospinal fluid (CSF). Th17 but also other CD4+ effector T-cell subsets could migrate from the basolateral to the apical side of the blood-cerebrospinal fluid barrier (BCSFB) in vitro, however, diapedesis of effector Th cells including that of Th17 cells did not require interaction of CCR6 with BCSFB derived CCL20. Our data underscore the important role of the ChP as CNS immune cell entry site in the context of autoimmune neuroinflammation.

Reduction of inflammation and mitochondrial degeneration in mutant SOD1 mice through inhibition of voltage-gated potassium channel Kv1.3.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no effective therapy, causing progressive loss of motor neurons in the spinal cord, brainstem, and motor cortex. Regardless of its genetic or sporadic origin, there is currently no cure for ALS or therapy that can reverse or control its progression. In the present study, taking advantage of a human superoxide dismutase-1 mutant (hSOD1-G93A) mouse that recapitulates key pathological features of human ALS, we investigated the possible role of voltage-gated potassium channel Kv1.3 in disease progression. We found that chronic administration of the brain-penetrant Kv1.3 inhibitor, PAP-1 (40 mg/Kg), in early symptomatic mice (i) improves motor deficits and prolongs survival of diseased mice (ii) reduces astrocyte reactivity, microglial Kv1.3 expression, and serum pro-inflammatory soluble factors (iii) improves structural mitochondrial deficits in motor neuron mitochondria (iv) restores mitochondrial respiratory dysfunction. Taken together, these findings underscore the potential significance of Kv1.3 activity as a contributing factor to the metabolic disturbances observed in ALS. Consequently, targeting Kv1.3 presents a promising avenue for modulating disease progression, shedding new light on potential therapeutic strategies for ALS.