Spatiotemporal progression of ubiquitin-proteasome system inhibition after status epilepticus suggests protective adaptation against hippocampal injury.

BACKGROUND: The ubiquitin-proteasome-system (UPS) is the major intracellular pathway leading to the degradation of unwanted and/or misfolded soluble proteins. This includes proteins regulating cellular survival, synaptic plasticity and neurotransmitter signaling; processes controlling excitability thresholds that are altered by epileptogenic insults. Dysfunction of the UPS has been reported to occur in a brain region- and cell-specific manner and contribute to disease progression in acute and chronic brain diseases. Prolonged seizures, status epilepticus, may alter UPS function but there has been no systematic attempt to map when and where this occurs in vivo or to determine the consequences of proteasome inhibition on seizure-induced brain injury. METHOD: To determine whether seizures lead to an impairment of the UPS, we used a mouse model of status epilepticus whereby seizures are triggered by an intra-amygdala injection of kainic acid. Status epilepticus in this model causes cell death in selected brain areas, in particular the ipsilateral CA3 subfield of the hippocampus, and the development of epilepsy after a short latent period. To monitor seizure-induced dysfunction of the UPS we used a UPS inhibition reporter mouse expressing the ubiquitin fusion degradation substrate ubiquitin(G76V)-green fluorescent protein. Treatment with the specific proteasome inhibitor epoxomicin was used to establish the impact of proteasome inhibition on seizure-induced pathology. RESULTS AND CONCLUSIONS: Our studies show that status epilepticus induced by intra-amygdala kainic acid causes select spatio-temporal UPS inhibition which is most evident in damage-resistant regions of the hippocampus, including CA1 pyramidal and dentate granule neurons then appears later in astrocytes. In support of this exerting a beneficial effect, injection of mice with the proteasome inhibitor epoxomicin protected the normally vulnerable hippocampal CA3 subfield from seizure-induced neuronal death in the model. These studies reveal brain region- and cell-specific UPS impairment occurs after seizures and suggest UPS inhibition can protect against seizure-induced brain damage. Identifying networks or pathways regulated through the proteasome after seizures may yield novel target genes for the treatment of seizure-induced cell death and possibly epilepsy

Application of high-throughput qPCR analysis in vaccine candidate's against Leishmania infantum: a practical case for BALB/c mice immunised with rLiPABP3

2 p.Peer reviewe

The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model

The interaction of Leishmaniawith BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver. . .) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation. The development of technical platforms for high-throughputqPCR analysis, and powerful software for analysis of qPCR data, have acknowledged the problem that some reference genes widely used due to their known or suspected “housekeeping” roles, should be avoided due to high expression variability across different tissues or experimental conditions. In this paper we evaluated the stability of 112 genes using three different algorithms: geNorm, NormFinder and RefFinder in spleen samples from BALB/c mice under different experimental conditions (control andLeishmania infantum-infected mice). Despite minor discrepancies in the stability ranking shown by the three methods, most genes show very similar performance as RG (either good or poor) across this massive data set. Our results show that some of the genes traditionally used as RG in this model (i.e.B2m,Polr2aandTbp) are clearly outperformed by others. In particular, the combination ofIl2rg+Itgb2was identified among the best scoring candidate RG for every group of mice and every algorithm used in this experimental model. Finally, we have demonstrated that using “traditional” vs rationally-selected RG for normalization of gene expression data may lead to loss of statistical significance of gene expression changes when using large-scale platforms, and therefore misinterpretation of results. Taken together, our results highlight the need for a comprehensive, high-throughput search for the most stable reference genes in each particularexperimental model.Revisión por pare

Spatiotemporal progression of ubiquitin-proteasome system inhibition after status epilepticus suggests protective adaptation against hippocampal injury

Background: The ubiquitin-proteasome-system (UPS) is the major intracellular pathway leading to the degradation of unwanted and/or misfolded soluble proteins. This includes proteins regulating cellular survival, synaptic plasticity and neurotransmitter signaling; processes controlling excitability thresholds that are altered by epileptogenic insults. Dysfunction of the UPS has been reported to occur in a brain region- and cell-specific manner and contribute to disease progression in acute and chronic brain diseases. Prolonged seizures, status epilepticus, may alter UPS function but there has been no systematic attempt to map when and where this occurs in vivo or to determine the consequences of proteasome inhibition on seizure-induced brain injury. Method: To determine whether seizures lead to an impairment of the UPS, we used a mouse model of status epilepticus whereby seizures are triggered by an intra-amygdala injection of kainic acid. Status epilepticus in this model causes cell death in selected brain areas, in particular the ipsilateral CA3 subfield of the hippocampus, and the development of epilepsy after a short latent period. To monitor seizure-induced dysfunction of the UPS we used a UPS inhibition reporter mouse expressing the ubiquitin fusion degradation substrate ubiquitin-green fluorescent protein. Treatment with the specific proteasome inhibitor epoxomicin was used to establish the impact of proteasome inhibition on seizure-induced pathology. Results and conclusions: Our studies show that status epilepticus induced by intra-amygdala kainic acid causes select spatio-temporal UPS inhibition which is most evident in damage-resistant regions of the hippocampus, including CA1 pyramidal and dentate granule neurons then appears later in astrocytes. In support of this exerting a beneficial effect, injection of mice with the proteasome inhibitor epoxomicin protected the normally vulnerable hippocampal CA3 subfield from seizure-induced neuronal death in the model. These studies reveal brain region- and cell-specific UPS impairment occurs after seizures and suggest UPS inhibition can protect against seizure-induced brain damage. Identifying networks or pathways regulated through the proteasome after seizures may yield novel target genes for the treatment of seizure-induced cell death and possibly epilepsy.Health Research Board (POR/2011/41 to D.C.H.), Science Foundation Ireland (13/IA/1891, 14/ADV/RC2721 and 12/COEN/18 to D.C.H. and 13/SIRG/2098 to T.E), EMBO (Short term fellowship ASTF No: 458–09 to TE), MSCA H2020 IF 707530 (to G.P.B.), Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CiberNed-Instituto de salud Carlos III) collaborative grant PI2013/09-2 to JJL, CoEN grant (NEURO-MIR to JJL) and by grants from Spanish Ministry of Economy and Competitiveness (MINECO, SAF2012-34177 and SAF2015-65371-R to JJL), Fundación BBVA and Fundación Ramón Arece

Interleukin-36 cytokines alter the intestinal microbiome and can protect against obesity and metabolic dysfunction

Members of the interleukin-1 (IL-1) family are important mediators of obesity and metabolic disease and have been described to often play opposing roles. Here we report that the interleukin-36 (IL-36) subfamily can play a protective role against the development of disease. Elevated IL-36 cytokine expression is found in the serum of obese patients and negatively correlates with blood glucose levels among those presenting with type 2 diabetes. Mice lacking IL-36Ra, an IL-36 family signalling antagonist, develop less diet-induced weight gain, hyperglycemia and insulin resistance. These protective effects correlate with increased abundance of the metabolically protective bacteria Akkermansia muciniphila in the intestinal microbiome. IL-36 cytokines promote its outgrowth as well as increased colonic mucus secretion. These findings identify a protective role for IL-36 cytokines in obesity and metabolic disease, adding to the current understanding of the role the broader IL-1 family plays in regulating disease pathogenesis.ISSN:2041-172

Ranking of top-ten reference genes in control and <i>Leishmania</i>-infected BALB/c.mice.

<p>Ranking of top-ten reference genes in control and <i>Leishmania</i>-infected BALB/c.mice.</p

Stability values of the best candidate reference genes (light gray) and 6 six classical reference genes (black bars) in spleen samples of control BALB/c mice.

<p><b>A)</b> M-stability value according to geNorm; horizontal line marks the threshold stability value M = 0.5. <b>B)</b> Pairwise variation (<i>V</i>_<i>n/n+1</i>) between the normalization factors of the samples according to geNorm. <b>C)</b> Stability ranking according to NormFinder. <b>D)</b> Stability ranking according to RefFinder. Lower values indicate higher stability for all rankings.</p

Ranking of top-ten reference genes in control BALB/c mice.

<p>Ranking of top-ten reference genes in control BALB/c mice.</p