Endoplasmic reticulum stress-mediated upregulation of miR-29a enhances sensitivity to neuronal apoptosis.

Disturbance of homeostasis within the endoplasmic reticulum (ER) lumen leads to the accumulation of unfolded and misfolded proteins. This results in the activation of an evolutionary conserved stress response termed ER stress that, if unresolved, induces apoptosis. Previously the Bcl-2 homology domain 3-Only Protein Puma was identified as a mediator of ER stress-induced apoptosis in neurons. In the search of alternative contributors to ER stress-induced apoptosis, a downregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 was noted during ER stress in both mouse cortical neurons and human SH-SY5Y neuroblastoma cells. Downregulation of Mcl-1 was associated with an upregulation of microRNA-29a (miR-29a) expression, and subsequent experiments showed that miR-29a targeted the 3'-untranslated region of the anti-apoptotic Bcl-2 family protein, Mcl-1. Inhibition of miR-29a expression using sequence-specific antagomirs or the overexpression of Mcl-1 decreased cell death following tunicamycin treatment, while gene silencing of Mcl-1 increased cell death. miR-29a did not alter the signalling branches of the ER stress response, rather its expression was controlled by the ER stress-induced transcription factor activating-transcription-factor-4 (ATF4). The current data demonstrate that the ATF4-mediated upregulation of miR-29a enhances the sensitivity of neurons to ER stress-induced apoptosis.

The APP intracellular domain (AICD) potentiates ER stress-induced apoptosis.

Here we employed human SHEP neuroblastoma cells either stably or inducibly expressing the amyloid precursor protein (APP) intracellular domain (AICD) to investigate its ability to modulate stress-induced cell death. Analysis of effector caspase activation revealed that AICD overexpression was specifically associated with an increased sensitivity to apoptosis induced by the 2 endoplasmic reticulum (ER) stressors thapsigargin and tunicamycin, but not by staurosporine (STS). Basal and ER stress-induced expression of Bip/Grp78 and C/EBP-homologous protein/GADD153 were not altered by AICD implying that AICD potentiated cell death downstream or independent of the conserved unfolded protein response (UPR). Interestingly, quantitative polymerase chain reaction analysis and reporter gene assays revealed that AICD significantly downregulated messenger RNA levels of the Alzheimer's disease susceptibility gene ApoJ/clusterin, indicating transcriptional repression. Knockdown of ApoJ/clusterin mimicked the effect of AICD on ER stress-induced apoptosis, but had no discernible effect on staurosporine-induced cell death. Our data suggest that altered levels of AICD may abolish the prosurvival function of ApoJ/clusterin and increase the susceptibility of neurons to ER stress-mediated cell death, a pathway that may contribute to the pathogenesis of Alzheimer's disease.

Prednisolone treatment induces tolerogenic dendritic cells and a regulatory milieu in myasthenia gravis patients.

FOXP3-expressing naturally occurring CD4(+)CD25(high) T regulatory cells (Treg) are relevant in the control of autoimmunity, and a defect in this cell population has been observed in several human autoimmune diseases. We hypothesized that altered functions of peripheral Treg cells might play a role in the immunopathogenesis of myasthenia gravis, a T cell-dependent autoimmune disease characterized by the presence of pathogenic autoantibodies specific for the nicotinic acetylcholine receptor. We report in this study a significant decrease in the in vitro suppressive function of peripheral Treg cells isolated from myasthenia patients in comparison to those from healthy donors. Interestingly, Treg cells from prednisolone-treated myasthenia gravis patients showed an improved suppressive function compared with untreated patients, suggesting that prednisolone may play a role in the control of the peripheral regulatory network. Indeed, prednisolone treatment prevents LPS-induced maturation of monocyte-derived dendritic cells by hampering the up-regulation of costimulatory molecules and by limiting secretion of IL-12 and IL-23, and enhancing IL-10. In addition, CD4(+) T cells cultured in the presence of such tolerogenic dendritic cells are hyporesponsive and can suppress autologous CD4(+) T cell proliferation. The results shown in this study indicate that prednisolone treatment promotes an environment that favors immune regulation rather than inflammation.

Interferon-gamma regulates cathepsin G activity in microglia-derived lysosomes and controls the proteolytic processing of myelin basic protein in vitro.

The serine protease cathepsin (Cat) G dominates the proteolytic processing of the multiple sclerosis (MS)-associated autoantigen myelin basic protein (MBP) in lysosomes from primary human B cells and dendritic cells. This is in contrast to B-lymphoblastoid cell lines, where the asparagine endopeptidase (AEP) is responsible for this task. We have analysed microglia-derived lysosomal proteases for their ability to process MBP in vitro. In lysosomes derived from primary murine microglia, CatD, CatS, AEP and CatG were involved in the processing of MBP. Interestingly, when microglia were treated with interferon-gamma to mimic a T helper type 1-biased cytokine milieu in MS, CatG was drastically down-regulated, in contrast to CatS, CatB, CatL, CatD or AEP. This resulted in significantly increased stability of MBP and a selective lack of CatG-derived proteolytic fragments; however, it did not affect the gross pattern of MBP processing. Inhibition of serine proteases eliminated the processing differences between lysosomal extracts from resting microglia compared to interferon-stimulated microglia. Thus, the cytokine environment modulates lysosomal proteases in microglia by a selective down-regulation of CatG, leading to decreased MBP-processing by microglia-derived lysosomal proteases in vitro.