Physiopathological Protein Release by Glial Cells: Focus on Purine Nucleoside Phosphorylase (PNP), Superoxide Dismutase 1 (SOD1) and α-Synuclein.

The interest on Purine Nucleoside Phosphorylase (PNP), Superoxide Dismutase 1 (SOD1) and α-synuclein derived from the fact that they are all involved in brain neuropathology.Mutations in the gene codifying for PNP induce PNP deficiency, a neurodevelopmental disorder characterized by immunodeficiency; SOD1 and alpha-synuclein are involved in amyotrophic lateral sclerosis and Parkison’s disease respectively, both characterized by the presence of protein aggregates, neurodegeneration and neuroinflammation. Because in all these neurological diseases there is a strong involvement of immune cells, we focused on the role of glial cell secretion. Here we obtained data on PNP release by glial cells and we could speculate that, by releasing this enzyme, these cells may support neuronal activity, by maintaining the homeostasis of the purinergic system. In particular, since cerebellar neurons displayed a low content and reduced capability of releasing PNP, we hypothesize that glial PNP is particularly important for their function. By using in vitro models of rat primary cultures of glial cells overexpressing wt or mutated SOD1, we observed that SOD1 carrying the mutations linked to fALS is less released than wild-type and accumulates intracellularly promoting the autophagy impairment and in turn, glial activation that might be responsible for reduced neuroprotection against glutamate excitotoxicity in co-cultures with cerebellar granule neurons. Trehalose seems to restore the physiological expression and release of mutant SOD1, as well as the physiological neuroprotective role of microglia. Astrocytes seem to be less susceptible to overexpression of mutated SOD1. α-synuclein carrying the mutations linked to familial forms of PD is more released than wild-type, in accordance to the increased release promoted by activated glial cells. The increased toxicity due to overexpression of mutant α-synuclein together with the increased release might be responsible for reduced neuroprotection and for the diffusion of potential toxic molecules support the prion-like hypothesis for Parkinson’s Disease

New models of Parkinson’s like neuroinflammation in human microglia clone 3: Activation profiles induced by INF-γ plus high glucose and mitochondrial inhibitors

Microglia activation and neuroinflammation have been extensively studied in murine models of neurodegenerative diseases; however, to overcome the genetic differences between species, a human cell model of microglia able to recapitulate the activation profiles described in patients is needed. Here we developed human models of Parkinson’s like neuroinflammation by using the human microglia clone 3 (HMC3) cells, whose activation profile in response to classic inflammatory stimuli has been controversial and reported only at mRNA levels so far. In fact, we showed the increased expression of the pro-inflammatory markers iNOS, Caspase 1, IL-1β, in response to IFN-γ plus high glucose, a non-specific disease stimulus that emphasized the dynamic polarization and heterogenicity of the microglial population. More specifically, we demonstrated the polarization of HMC3 cells through the upregulation of iNOS expression and nitrite production in response to the Parkinson’s like stimuli, 6-hydroxidopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the latter depending on the NF-κB pathway. Furthermore, we identified inflammatory mediators that promote the pro-inflammatory activation of human microglia as function of different pathways that can simulate the phenotypic transition according to the stage of the pathology. In conclusion, we established and characterized different systems of HMC3 cells activation as in vitro models of Parkinson’s like neuroinflammation

In vivo morphological alterations of TAMs during KCa3.1 inhibition—by using in vivo two-photon time-lapse technology

Tumor associated macrophages (TAMs) are the mostprevalent cells recruited in the tumor microenvironment (TME). Once recruited, TAMs acquire a pro-tumor phenotype characterized by a typical morphology: ameboid in the tumor core and with larger soma and thick branches in the tumor periphery. Targeting TAMs by reverting them to an anti-tumor phenotype is a promising strategy for cancer immunotherapy. Taking advantage of Cx3cr1GFP/WT heterozygous mice implanted with murine glioma GL261-RFP cells we investigated the role of Ca2+-activated K+ channel (KCa3.1) on the phenotypic shift of TAMs at the late stage of glioma growth through in vivo two-photon imaging. We demonstrated that TAMs respond promptly to KCa3.1 inhibition using a selective inhibitor of the channel (TRAM-34) in a time-dependent manner by boosting ramified projections attributable to a less hypertrophic phenotype in the tumor core. We also revealed a selective effect of drug treatment by reducing both glioma cells and TAMs in the tumor core with no interference with surrounding cells. Taken together, our data indicate a TRAM-34-dependent progressive morphological transformation of TAMs toward a ramified and anti-tumor phenotype, suggesting that the timing of KCa3.1 inhibition is a key point to allow beneficial effects on TAMs

Histoire et anthropologie sociale de l’Italie moderne et contemporaine

Gérard Delille, Daniel Fabre, Pierre-Antoine Fabre, directeurs d’étudesMartine Boiteux, professeur agrégéeJean-François Chauvard, directeur des études à l’École française de RomeMaria Antonietta Visceglia, professeur à l’Université de Rome-La SapienzaMarcello Massenzio, professeur à l’Université Rome-IIFrancesca Cantù, professeur à l’Université Rome-III Langues de pouvoir. Pouvoir des langues Le séminaire se déroule désormais suivant la nouvelle formule mise en place en 2006-2007 qui permet a..

Targeted quantitative metabolic profiling of brain-derived cell cultures by semi-automated MEPS and LC-MS/MS

The accurate characterisation of metabolic profiles is an important prerequisite to determine the rate and the efficiency of the metabolic pathways taking place in the cells. Changes in the balance of metabolites involved in vital processes such as glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), as well as in the biochemical pathways related to amino acids, lipids, nucleotides, and their precursors reflect the physiological condition of the cells and may contribute to the development of various human diseases. The feasible and reliable measurement of a wide array of metabolites and biomarkers possesses great potential to elucidate physiological and pathological mechanisms, aid preclinical drug development and highlight potential therapeutic targets. An effective, straightforward, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was developed for the simultaneous quali-quantitative analysis of 41 compounds in both cell pellet and cell growth medium obtained from brain-derived cell cultures. Sample pretreatment miniaturisation was achieved thanks to the development and optimisation of an original extraction/purification approach based on digitally programmed microextraction by packed sorbent (eVol®-MEPS). MEPS allows satisfactory and reproducible clean-up and preconcentration of both low-volume homogenate cell pellet lysate and cell growth medium with advantages including, but not limited to, minimal sample handling and method sustainability in terms of sample, solvents, and energy consumption. The MEPS-LC-MS/MS method showed good sensitivity, selectivity, linearity, and precision. As a proof of concept, the developed method was successfully applied to the analysis of both cell pellet and cell growth medium obtained from a line of mouse immortalised oligodendrocyte precursor cells (OPCs; Oli-neu cell line), leading to the unambiguous determination of all the considered target analytes. This method is thus expected to be suitable for targeted, quantitative metabolic profiling in most brain cell models, thus allowing accurate investigations on the biochemical pathways that can be altered in central nervous system (CNS) neuropathologies, including e.g., mitochondrial respiration and glycolysis, or use of specific nutrients for growth and proliferation, or lipid, amino acid and nucleotide metabolism

Histoire et anthropologie : politique, société, culture, religion

Gérard Delille, Daniel Fabre, Pierre-Antoine Fabre, directeurs d’étudesMartine Boiteux, professeur agrégéeStefano Andretta, Francesca Cantù, professeurs à l’Université Rome-IIIJean-François Chauvard, directeur d’études à l’École française de RomeMarcello Massenzio, professeur à l’Université Rome Tor-VergataAntonio Prosperi, professeur à l’ENS de PiseMauro Ronzani, professeur à l’Université de PiseMaria Antonietta Visceglia, professeur à l’Université de Rome La Sapienza Temps du mythe/temps de..

PLAC1 immunization does not induce infertility in mice

Aim: Placenta specific 1 (PLAC1) is a protein rarely expressed in normal cells, except it is important for placental development, with a possible role in the establishment of the mother-fetus interface. The gene is also highly active in a wide variety of cancers and therefore, immunization with PLAC1 peptides could possibly be part of future immunotherapeutic strategies. We investigated whether vaccination against PLAC1 could induce infertility. Materials & methods: We inoculated female mice with PLAC1 peptides, put them in mating, measured antibody response (ELISA assay) and checked, in immunohistochemistry, binding of the induced antibodies to the native antigen. Results: We demonstrated that mice consistently develop antibody responses. We also demonstrated that female mice, after being inoculated with the PLAC1 peptide mix, do became pregnant and can give birth to normal infants. Conclusion: PLAC1 antigens as a specific anti-cancer vaccine could induce anti-PLAC1 antibodies which do not necessarily cause infertility

Selection of suitable reference genes for gene expression studies in HMC3 cell line by quantitative real-time RT-PCR

Abstract Microglia represent the primary immune defense system within the central nervous system and play a role in the inflammatory processes occurring in numerous disorders, such as Parkinson’s disease (PD). PD onset and progression are associated with factors considered possible causes of neuroinflammation, i.e. genetic mutations. In vitro models of microglial cells were established to identify specific molecular targets in PD through the analysis of gene expression data. Recently, the Human Microglial Clone 3 cell line (HMC3) has been characterized and a new human microglia model has emerged. Here we perform RT-qPCR analyses to evaluate the expression of ten reference genes in HMC3, untreated or stimulated to a pro-inflammatory status. The comparative ∆CT method, BestKeeper, Normfinder, geNorm and RefFinder algorithms were used to assess the stability of the candidate genes. The results showed that the most suitable internal controls are HPRT1, RPS18 and B2M genes. In addition, the most stable and unstable reference genes were used to normalize the expression of a gene of interest in HMC3, resulting in a difference in the statistical significance in cells treated with Rotenone. This is the first reference gene validation study in HMC3 cell line in pro-inflammatory status and can contribute to more reliable gene expression analysis in the field of neurodegenerative and neuroinflammatory research

Nutritional and Pharmacological Strategies to Regulate Microglial Polarization in Cognitive Aging and Alzheimer’s Disease

The study of microglia, the immune cells of the brain, has experienced a renaissance after the discovery of microglia polarization. In fact, the concept that activated microglia can shift into the M1 pro-inflammatory or M2 neuroprotective phenotypes, depending on brain microenvironment, has completely changed the understanding of microglia in brain aging and neurodegenerative diseases. Microglia polarization is particularly important in aging since an increased inflammatory status of body compartments, including the brain, has been reported in elderly people. In addition, inflammatory markers, mainly derived from activated microglia, are widely present in neurodegenerative diseases. Microglial inflammatory dysfunction, also linked to microglial senescence, has been extensively demonstrated and associated with cognitive impairment in neuropathological conditions related to aging. In fact, microglia polarization is known to influence cognitive function and has therefore become a main player in neurodegenerative diseases leading to dementia. As the life span of human beings increases, so does the prevalence of cognitive dysfunction. Thus, therapeutic strategies aimed to modify microglia polarization are currently being developed. Pharmacological approaches able to shift microglia from M1 pro-inflammatory to M2 neuroprotective phenotype are actually being studied, by acting on many different molecular targets, such as glycogen synthase kinase-3 (GSK3) β, AMP-activated protein kinase (AMPK), histone deacetylases (HDACs), etc. Furthermore, nutritional approaches can also modify microglia polarization and, consequently, impact cognitive function. Several bioactive compounds normally present in foods, such as polyphenols, can have anti-inflammatory effects on microglia. Both pharmacological and nutritional approaches seem to be promising, but still need further development. Here we review recent data on these approaches and propose that their combination could have a synergistic effect to counteract cognitive aging impairment and Alzheimer’s disease (AD) through immunomodulation of microglia polarization, i.e., by driving the shift of activated microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype