Immunogenicity of neuroblastoma tumors is controlled by impaired activity of NF-kB and IRF1 transcription factors.

La maggior parte dei tumori riescono ad evadere il sistema immunitario inibendo l’espressione di antigeni tumorali associati alle molecole del complesso maggiore di istocompatibilità di classe I (MHC I) sulla superficie cellulare. La mancata espressione di questi complessi è spesso dovuta alla presenza di difetti strutturali dei geni codificanti le molecole MHC I, oppure all’aberrante espressione delle molecole responsabili del processamento degli antigeni legati alle molecole MHC I. Il neuroblastoma (NB), il tumore extracraniale solido più comune dell’infanzia, non è un’eccezione. Sia la maggior parte delle linee cellulari di NB, che i tumori primari esprimono bassi, se non nulli, livelli di MHC I che possono essere aumentati trattando le cellule con l’interferone-gamma (IFN-γ). Questo fenotipo è compatibile con la presenza di difetti nella regolazione trascrizionale delle molecole coinvolte nel processamento e nella presentazione dell’antigene. Il presente studio ha lo scopo di indagare il meccanismo molecolare che determina la mancata o ridotta espressione delle molecole MHC I, e delle due aminopeptidasi del reticolo endoplasmatico ERAP1 ed ERAP2. Le forme più aggressive di NB sono caratterizzate dall’amplificazione dell’oncogene MYCN. Sebbene una correlazione inversa tra l’espressione di MYCN le molecole MHC I nelle linee cellulari umane di NB sia stata riportata, un coinvolgimento diretto di MYCN nella regolazione di MHC I non è stato dimostrato. I nostri risultati dimostrano che MYCN non è responsabile dei bassi livelli di MHC I, ERAP1 ed ERAP2 nelle cellule di NB analizzate, infatti la loro espressione non è influenzata nè dalla forzata espressione ne dall’inibizione di MYCN. Abbiamo invece identificato due fattori di trascrizione, NF-kB e IRF1, che sono direttamente coinvolti nella regolazione delle proteine MHC I ed ERAPs. Mediante il saggio di immunoprecipitazione della cromatina abbiamo dimostrato che il reclutamento di p65 (una subunità di NF-kB) sui promotori di MHC I, ERAP1 ed ERAP2 è direttamente proporzionale all’espressione di questi geni. Inoltre, il fenotipo negativo per MHC I, ERAP1 ed ERAP2, caratteristico delle forme più aggressive di NB, coincide con una bassa attività nucleare di NF-kB ed IRF1. L’overespressione dei due fattori trascrizionali da soli è in grado di recuperare solo parzialmente l’espressione di MHC I, ERAP1 ed ERAP2, inoltre il risultato dipende dalla linea cellulare trasfettata. Comunque, la trasfezione contemporanea di NF-kB ed IRF1 produce un incremento sinergico dei geni target in tutte le linee trasfettate. Degno di nota è il fatto che l’espressione di p65 nei tumori primari di neuroblastoma è simile a quella osservata nelle linee cellulari. Infatti, solo le cellule gangliari, ovvero le cellule più differenziate presenti nel tessuto tumorale, esprimono sia l’MHC I che il p65 nucleare. Quindi, questo studio mette in luce il meccanismo molecolare responsabile della mancata espressione delle molecole MHC I, ERAP1 ed ERAP2 nei tumori di neuroblastoma più aggressivi fornendo un importante punto di partenza per lo sviluppo di protocolli immunoterapeutici più efficaci basati sull’utilizzo delle cellule T.Low expression of major histocompatibility complex class I (MHC I) molecules on the cell surface allows tumors to evade the host T cell-based immune response. These abnormalities are often related to either genetic defects of MHC I genes or aberrant expression of antigen processing machinery (APM) components. Neuroblastoma (NB), the most common solid extracranial cancer of childhood, is not an exception. MHC I surface expression is virtually undetectable in the most NB cell lines and primary tumors, and upregulated by gamma-interferon (IFN-γ). This phenotype is compatible with defects in the regulation of antigen processing and presentation components. In this study, the molecular mechanism underlying low immunogenicity in neuroblastoma was investigated. Amplification of the MYCN oncogene characterizes the most aggressive forms of NB and is believe to downregulate expression of MHC class I molecules. Although an inverse correlation between MYCN and MHC I has been reported in human NB cell lines, a direct demonstration of the MYCN-mediated down-regulation of MHC I expression has been questioned. Herein, we demonstrate that MYCN is not responsible for low MHC I, ERAP1 and ERAP2 protein levels in human NB cell lines, since their expression is not affected by neither transfection-mediated overexpression nor siRNA suppression of MYCN. Instead, we identified NF-kB and IRF1 as the main factors involved in the transcriptional regulation of MHC I and ERAPs proteins. By chromatin immunoprecipitation assay, we show a recruitment of p65 NF-kB to the MHC I, ERAP1 and ERAP2 promoters that is proportional with the expression of these genes. Moreover, low nuclear activity of both NF-kB and IRF1 factors correlated with the MHC I, ERAP1 and ERAP2-low phenotype of the most aggressive NB cell lines. Overexpression of either the transcription factors alone rescued the MHC I, ERAP1 and ERAP2-low phenotype, but only partially and in a cell-type depending manner. Important, the co-transfection of both NF-kB and IRF1 cooperated to strongly enhance the transactivation of MHC I, ERAP1 and ERAP2 in any cell lines. Notheworthy, NF-kB and IRF1 acted in a synergistic manner. We found an intriguing parallel in primary NB tumors, in fact, nuclear p65 was detected in the maturing neuroblastic cells (i.e. ganglionic cells) which express higher levels of MHC I molecules in human NB specimens. These findings provide molecular insight into defective MHC I expression in NB tumors and indicate that activating NF-kB and IRF1 in MHC I-low, aggressive NB cells could be instrumental for successful application of T cell-based immunotherapy

Induced pluripotent stem cell-based organ-on-a-chip as personalized drug screening tools: A focus on neurodegenerative disorders

The Organ-on-a-Chip (OoC) technology shows great potential to revolutionize the drugs development pipeline by mimicking the physiological environment and functions of human organs. The translational value of OoC is further enhanced when combined with patient-specific induced pluripotent stem cells (iPSCs) to develop more realistic disease models, paving the way for the development of a new generation of patient-on-a-chip devices. iPSCs differentiation capacity leads to invaluable improvements in personalized medicine. Moreover, the connection of single-OoC into multi-OoC or body-on-a-chip allows to investigate drug pharmacodynamic and pharmacokinetics through the study of multi-organs cross-talks. The need of a breakthrough thanks to this technology is particularly relevant within the field of neurodegenerative diseases, where the number of patients is increasing and the successful rate in drug discovery is worryingly low. In this review we discuss current iPSC-based OoC as drug screening models and their implication in development of new therapies for neurodegenerative disorders

On-going frontal alpha rhythms are dominant in passive state and desynchronize in active state in adult gray mouse lemurs

The gray mouse lemur (Microcebus murinus) is considered a useful primate model for translational research. In the framework of IMI PharmaCog project (Grant Agreement n°115009, www.pharmacog.org), we tested the hypothesis that spectral electroencephalographic (EEG) markers of motor and locomotor activity in gray mouse lemurs reflect typical movement-related desynchronization of alpha rhythms (about 8-12 Hz) in humans. To this aim, EEG (bipolar electrodes in frontal cortex) and electromyographic (EMG; bipolar electrodes sutured in neck muscles) data were recorded in 13 male adult (about 3 years) lemurs. Artifact-free EEG segments during active state (gross movements, exploratory movements or locomotor activity) and awake passive state (no sleep) were selected on the basis of instrumental measures of animal behavior, and were used as an input for EEG power density analysis. Results showed a clear peak of EEG power density at alpha range (7-9 Hz) during passive state. During active state, there was a reduction in alpha power density (8-12 Hz) and an increase of power density at slow frequencies (1-4 Hz). Relative EMG activity was related to EEG power density at 2-4 Hz (positive correlation) and at 8-12 Hz (negative correlation). These results suggest for the first time that the primate gray mouse lemurs and humans may share basic neurophysiologic mechanisms of synchronization of frontal alpha rhythms in awake passive state and their desynchronization during motor and locomotor activity. These EEG markers may be an ideal experimental model for translational basic (motor science) and applied (pharmacological and non-pharmacological interventions) research in Neurophysiology

Chemical exchange saturation transfer MRI shows low cerebral 2-deoxy-D-glucose uptake in a model of Alzheimer's Disease

Glucose is the central nervous system's only energy source. Imaging techniques capable to detect pathological alterations of the brain metabolism are useful in different diagnostic processes. Such techniques are also beneficial for assessing the evaluation efficacy of therapies in pre-clinical and clinical stages of diseases. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a possible alternative to positron emission tomography (PET) imaging that has been widely explored in cancer research in humans and animal models. We propose that pathological alterations in brain 2-deoxy-D-glucose (2DG) uptake, typical of neurodegenerative diseases, can be detected with CEST MRI. Transgenic mice overexpressing a mutated form of amyloid precusrsor protein (APP23), a model of Alzheimer's disease, analyzed with CEST MRI showed a clear reduction of 2DG uptake in different brain regions. This was reminiscent of the cerebral condition observed in Alzheimer's patients. The results indicate the feasibility of CEST for analyzing the brain metabolic state, with better image resolution than PET in experimental models

Sporadic human prion diseases: molecular insights and diagnosis

Human prion diseases can be sporadic, inherited, or acquired by infection. Distinct clinical and pathological characteristics separate sporadic diseases into three phenotypes: Creutzfeldt-Jakob disease (CJD), fatal insomnia, and variably protease-sensitive prionopathy. CJD accounts for more than 90% of all cases of sporadic prion disease; it is commonly categorised into five subtypes that can be distinguished according to leading clinical signs, histological lesions, and molecular traits of the pathogenic prion protein. Three subtypes affect prominently cognitive functions whereas the other two impair cerebellar motor activities. An accurate and timely diagnosis depends on careful clinical examination and early performance and interpretation of diagnostic tests, including electroencephalography, quantitative assessment of the surrogate markers 14-3-3, tau, and of the prion protein in the CSF, and neuroimaging. The reliability of CSF tests is improved when these tests are interpreted alongside neuroimaging data

A minimal promoter for TFIIIC-dependent in vitro transcription of snoRNA and tRNA genes by RNA polymerase III.

The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA(Asn)(GTT) gene family, but not for two tRNA(Pro)(AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA(Asn) genes, but only in the presence of TFIIIB reconstituted with a crude B'' fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA(Asn) genes was strongly influenced by their 5'-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIIC-interacting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription

EU project UPGRADE (No 724036) - measurements of a Jeep Renegade prototype vehicle by JRC

Within Work Package 5 of the H2020 project UPGRADE (High efficient Particulate free Gasoline Engines) http://www.upgrade-project.eu/, JRC tested a demonstrator vehicle specifically developed by Centro Ricerche Fiat (CRF) in WLTP conditions. The results showed that: 1. All pollutants were below the EURO 6 limits. 2. Particle number emissions were two orders of magnitude below the limit of 6*10^11/km. PN10 were in average 13% higher than PN23. 3. CO2 emissions were on average 165.3 g/km.JRC.C.4-Sustainable Transpor

The Parkinson's disease-related protein DJ-1 protects dopaminergic neurons in vivo and cultured cells from alpha-synuclein and 6-hydroxydopamine toxicity

BACKGROUND Dopaminergic degeneration is a major finding in brains of patients with Parkinson's disease (PD), together with Lewy bodies, intraneuronal inclusions mainly composed of the fibrillogenic protein \textgreeka-synuclein (\textgreeka-syn). The familial-PD-related protein DJ-1 was reported to reduce dopaminergic degeneration triggered by \textgreeka-syn or by the dopaminergic-selective neurotoxin 6-hydroxydopamine (6-OHDA). OBJECTIVE The aim was to further investigate the role of DJ-1 in dopaminergic degeneration and to see whether a cell-permeable recombinant form of DJ-1 (TAT-DJ-1) could restore dopamine depletion in vivo, thus representing an innovative therapeutic approach. METHODS We developed in vitro (PC12/TetOn cells and mouse primary mesencephalic neurons) and in vivo models including DJ-1 knockout (-/-) mice to investigate DJ-1 in dopaminergic degeneration. RESULTS We found that in PC12/TetOn cells overexpressing \textgreek{a}-syn with the familial-PD linked mutation A30P, DJ-1 silencing increased \textgreek{a}-syn (A30P) toxicity. Primary mesencephalic neurons from DJ-1 (-/-) mice were more vulnerable to a cell-permeable form of \textgreek{a}-syn (TAT-\textgreek{a}-syn) and to 6-OHDA. Intrastriatally administered TAT-DJ-1 reduced 6-OHDA toxicity in vivo in C57BL/6 mice. Finally, when we injected TAT-\textgreek{a}-syn (A30P) in the striatum of DJ-1 (-/-) animals, dopamine was depleted more than in the control strain. CONCLUSION DJ-1 appears to have a protective role against dopaminergic degeneration triggered by \textgreek{a}-syn or 6-OHDA, reinforcing the possible therapeutic importance of this protein in PD

Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases