Strategies and tools for studying microglial-mediated synapse elimination and refinement

The role of microglia in controlling synapse homeostasis is becoming increasingly recognized by the scientific community. In particular, the microglia-mediated elimination of supernumerary synapses during development lays the basis for the correct formation of neuronal circuits in adulthood, while the possible reactivation of this process in pathological conditions, such as schizophrenia or Alzheimer\u27s Disease, provides a promising target for future therapeutic strategies. The methodological approaches to investigate microglial synaptic engulfment include differen

new role of atm in controlling gabaergic tone during development

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Regulation of peripheral T cell activation by calreticulin

Regulated expression of positive and negative regulatory factors controls the extent and duration of T cell adaptive immune response preserving the organism's integrity. Calreticulin (CRT) is a major Ca2+ buffering chaperone in the lumen of the endoplasmic reticulum. Here we investigated the impact of CRT deficiency on T cell function in immunodeficient mice reconstituted with fetal liver crt−/− hemopoietic progenitors. These chimeric mice displayed severe immunopathological traits, which correlated with a lower threshold of T cell receptor (TCR) activation and exaggerated peripheral T cell response to antigen with enhanced secretion of inflammatory cytokines. In crt−/− T cells TCR stimulation induced pulsatile cytosolic elevations of Ca2+ concentration and protracted accumulation of nuclear factor of activated T cells in the nucleus as well as sustained activation of the mitogen-activated protein kinase pathways. These observations support the hypothesis that CRT-dependent shaping of Ca2+ signaling critically contributes to the modulation of the T cell adaptive immune response

Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

Actin-based remodeling underlines spine morphogenesis and plasticity and is crucially involved in the processes that constantly reshape the circuitry of the adult brain in response to external stimuli, leading to learning and memory formation and supporting cognitive functions. Hence spine morphology and synaptic strength are tightly linked and indeed abnormalities in spine number and morphology have been described in a number of neurological disorders such as autism spectrum disorders (ASDs), schizophrenia and intellectual disabilities. We have recently demonstrated that the actin regulating protein, Epidermal growth factor receptor pathway substrate 8 (Eps8), is essential for spine growth and long term potentiation. Indeed, mice lacking Eps8 display immature filopodia-like spines, which are unable to undergo potentiation, and are impaired in cognitive functions. Furthermore, reduced levels of Eps8 have been found in the brain of a cohort of patients affected by ASD compared to controls. Here we investigated whether the lack of Eps8, which is also part of the N-methyl-d-aspartate (NMDA) receptor complex, affects the functional maturation of the postsynaptic compartment. Our results demonstrate that Eps8 knock out mice (Eps8 KO) neurons display altered synaptic expression and subunit composition of NMDA receptors (i.e., increased GluN2B-, decreased GluN2A-containing receptors) and impaired GluN2B to GluN2A subunit shift. Indeed Eps8 KO neurons display increased content of GluN2B containing NMDA receptors both at the synaptic and extrasynaptic level. Furthermore, Eps8 KO neurons display an increased content of extra-synaptic GluN2B-containing receptors, suggesting that also the synaptic targeting of NMDA receptors is affected by the lack of Eps8. These data demonstrate that, besides regulation of spine morphogenesis, Eps8 also regulates the synaptic balance of NMDA receptors subunits GluN2A and GluN2B

Acid sphingomyelinase activity triggers microparticle release from glial cells

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases

Ectonucleotidase activity and immunosuppression in astrocyte-CD4 T cell bidirectional signaling

Astrocytes play a crucial role in neuroinflammation as part of the glia limitans, which regulates infiltration of the brain parenchyma by leukocytes. The signaling pathways and molecular events, which result from the interaction of activated T cells with astrocytes are poorly defined. Here we show that astrocytes promote the expression and enzymatic activity of CD39 and CD73 ectonucleotidases in recently activated CD4 cells by a contact dependent mechanism that is independent of T cell receptor interaction with class II major histocompatibility complex (MHC). Transforming growth factor-β (TGF-β) is robustly upregulated and sufficient to promote ectonucleotidases expression. T cell adhesion to astrocyte results in differentiation to an immunosuppressive phenotype defined by expression of the transcription factor Rorγt, which characterizes the CD4 T helper 17 subset. CD39 activity in T cells in turn inhibits spontaneous calcium oscillations in astrocytes that correlated with enhanced and reduced transcription of CCL2 chemokine and Sonic hedgehog (Shh), respectively. We hypothesize this TCR-independent interaction promote an immunosuppressive program in T cells to control possible brain injury by deregulated T cell activation during neuroinflammation. On the other hand, the increased secretion of CCL2 with concomitant reduction of Shh might promote leukocytes extravasation into the brain parenchyma

Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients

The lack of biomarkers in Amyotrophic Lateral Sclerosis (ALS) makes it difficult to determine the stage of the disease in patients and, therefore, it delays therapeutic trials. Microvesicles (MVs) are possible biomarkers implicated in physiological and pathological functions, however, their role in ALS remains unclear. We investigated whether plasma derived microvesicles could be overrepresented in a group of 40 patients affected by ALS compared to 28 Alzheimer’s Disease (AD) patients and 36 healthy volunteers. Leukocyte derived MVs (LMVs) compared to endothelial, platelet, erythrocyte derived MVs, were mostly present in ALS patients compared to AD patients and healthy donors. Correlation analysis corrected for the presence of confounding variables (riluzole, age at onset, site of onset, gender) was tested between PRL (Progression Rate at the Last visit) and LMVs, and a statistically significant value was found (Pearson partial correlation r = 0.407, p = 0.006). We also investigated SOD1, TDP-43 intravesicular protein level in LMVs. Misfolded SOD1 was selectively transported by LMVs and its protein level was associated with the percentage of LMVs in slow progressing patients (r = 0.545, p = 0.033). Our preliminary findings suggest that LMVs are upregulated in ALS patients and they can be considered possible markers of disease progression