Congestive heart failure in COX2 deficient rats

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) formation by targeting cyclooxygenase (COX) 1 and 2.Long-term use of NSAIDs that selectively inhibit COX2 increases the risk for thrombotic events,cardiac failure,and hypertension.However,the underlying mechanisms remain unclear.In this study,COX1- and COX2-deficient rats were created via Cas9/RNA-mediated gene targeting.DNA genotyping and Western blot analysis confirmed successful generation of COX1~(-/-)and COX2~(-/-)rats.Adult COX1~(-/-)rats grew normally,while more than 70%of COX2~(-/-)rats after wean died within 2 months.Echocardiography showed markedly reduced left ventricular ejection fraction and fractional shortening in adult COX2~(-/-)rats compared to those in wildtype (WT) controls.Histological analysis revealed accumulation of inflammatory cells and severe interstitial and perivascular fibrosis in COX2~(-/-)cardiac tissues.Moreover,cardiac ATP and acetyl-Co A production was dramatically decreased in COX2~(-/-)rats.Consistently,the expression of genes related to mitochondrial oxidation,such as those that encode for subunits of pyruvate dehydrogenase complex and acyl Co A dehydrogenases,were downregulated,while glycolytic hexokinase 1 (HK1) was upregulated in COX2~(-/-)heart tissues.These observations indicate that COX2-deficient rats developed spontaneously heart failure,likely as a result of dysregulated cardiac energy metabolism.     

Selective response of various brain cell types during neurodegeneration induced by mild impairment of oxidative metabolism

Age-related neurodegenerative diseases are characterized by selective neuron loss, glial activation, inflammation and abnormalities in oxidative metabolism. Thiamine deficiency (TD) is a model of neurodegeneration induced by impairment of oxidative metabolism. TD produces a time-dependent, selective neuronal death in specific brain regions, while other cell types are either activated or unaffected. TD-induced neurodegeneration occurs first in a small, well-defined brain region, the submedial thalamic nucleus (SmTN). This discrete localization permits careful analysis of the relationship between neuronal loss and the response of other cell types. The temporal analysis of the changes in the region in combination with the use of transgenic mice permits testing of proposed mechanisms of how the interaction of neurons with other cell types produces neurodegeneration. Loss of neurons and elevation in markers of neurodegeneration are accompanied by changes in microglia including increased redox active iron, the induction of nitric oxide synthase (NOS) and hemeoxygenase-1, a marker of oxidative stress. Endothelial cells also show changes in early stages of TD including induction of intracellular adhesion molecule-1 (ICAM-1) and endothelial NOS. The number of degranulating mast cells also increases in early stages of TD. Alterations in astrocytes and neutrophils occur at later stages of TD. Studies with transgenic knockouts indicate that the endothelial cell changes are particularly important. We hypothesize that TD-induced abnormalities in oxidative metabolism promote release of neuronal inflammatory signals that activate microglia, astrocytes and endothelial cells. Although at early stages the responses of non-neuronal cells may be neuroprotective, at late phases they lead to entry of peripheral inflammatory cells into the brain and promote neurodegeneration.     

SAP suppresses the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated disease of the central nervous system. Serum amyloid P component (SAP) is a highly conserved plasma protein named for its universal presence in amyloid deposits. Here we report that SAP-transgenic mice had unexpectedly attenuated EAE due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant, SAP-transgenic mice showed reduced spinal cord inflammation with lower severity of EAE attacks as compared with control C57BL/6 mice. However, in SAP-Knockout mice, the severity of EAE is enhanced. Adoptive transfer of Ag-restimulated T cells from wild type to SAP-transgenic mice, or transfer of SAP-transgenic Ag-restimulated T cells to control mice, induced milder EAE. T cells from MOG-primed SAP-transgenic mice showed weak proliferative responses. Furthermore, in SAP-transgenic mice, there is little infiltration of CD45-positive cells in the spinal cord. In vitro, SAP suppressed the secretion of interleukin-2 stimulated by P-selectin and blocked P-selectin binding to T cells. Moreover, SAP could change the affinity between α4-integrin and T cells. These data suggested that SAP could antagonize the development of the acute phase of inflammation accompanying EAE by modulating the function of P-selectin.     

Tricarboxylic acid cycle enzymes following thiamine deficiency

Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the alpha-ketoglutarate dehydrogenase complex (KGDHC) (-21.5%) and PDHC (-10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase (STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter brain function in neurodegenerative disorders.     

Interaction between ERK and GSK3beta mediates basic fibroblast growth factor-induced apoptosis in SK-N-MC neuroblastoma cells

The Ewing's sarcoma family of tumors (ESFT) includes Ewing's sarcoma (ES), Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor. Basic fibroblast growth factor (FGF2) suppresses the growth of ESFT cells and causes their apoptosis. The underlying mechanism is unclear. Using a human peripheral primitive neuroectodermal tumor cell line, SK-N-MC, we demonstrated FGF2 stimulated phosphorylation of ERK1 and ERK2 (pERK1/2) and GSK3beta (pGSK3beta(Tyr-216)), all of which were primarily retained in the cytoplasm. FGF2 promoted the association between ERK and pGSK3beta(Tyr-216). Inhibitors for GSK3beta (TDZD and LiCl) and ERK (PD98059) protected cells from FGF2-induced apoptosis. On the other hand, inhibitors of GSK3beta, but not PD98059 decreased ERK/pGSK3beta(Tyr-216) association and caused a nuclear translocation of pERK1/2. Similarly, expression of a kinase-deficient (K85R) GSK3beta or GSK3beta-small interfering RNA inhibited FGF2-regulated ERK/pGSK3beta(Tyr-216) association and translocated pERK to the nucleus. Both K85R GSK3beta and small interfering RNA offered protection against FGF2-induced cell death. In contrast, overexpression of wild-type GSK3beta sensitized cells to FGF2 cytotoxicity. Hydrogen peroxide and ethanol enhanced FGF2-stimulated pGSK3beta(Tyr-216), ERK/pGSK3beta(Tyr-216) association, and cytoplasmic retention of pERK1/2. As a result, they potentiated FGF2-induced cell death. Taken together, our results suggested that FGF2-induced accumulation of pERK1/2 in the cytoplasm is toxic for SK-N-MC cells. The formation of an ERK.GSK3beta complex retained pERK1/2 in the cytoplasm. In contrast, disruption of the ERK.GSK3beta complex resulted in nuclear translocation of pERK1/2 and offered protection.     

细胞因子与酒精性肝病

酒精性肝病(alcoholic liver disease,ALD)的发生发展过程与体内多种细胞因子有关,尤其是肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和转化生长因子-β(transforming growth factor-β,TGF-β)在调节肝细胞的凋亡过程中具有重要作用。TNF-α可引起肝细胞凋亡与炎症反应等,抗TNF-α治疗能明显减轻酒精引起的肝损害;TGF-β具有增加细胞外基质的合成和抑制细胞外基质降解的作用,TGF-β1升高与肝纤维化密切相关。细胞因子可能是防治酒精性肝病的有效分子靶点。     

dsRNA binding protein PACT/RAX in gene silencing,development and diseases

PACT （Protein kinase, interferon-inducible double stranded RNA dependent activator） and its murine ortholog RAX （PKR-associated protein X） were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase （PKR）. Endogenous PACT/RAX activates PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. PACT/RAX heterodimerized with PKR and activated it with its third motif in the absence of dsRNA. The activation of PKR leads to enhanced eIF2a phosphorylation followed by apoptosis or inhibition of growth. Besides the role of activating PKR, PACT is associated with a ~500 kDa complex that contains Dicer, hAgo2, and TRBP （TAR RNA binding protein） and it associates with Dicer to facilitate the production of small interfering RNA. PACT/RAX plays an important role in diverse physiological and pathological processes. Pact^-/- mice exhibit notable developmental abnormalities including microtia, with craniofacial ear, and hearing defects. Pact^-/- mice had smaller body sizes and fertility defects, both of which were caused by defective pituitary functions. It was found that dRAX disrupted fly embryos homozygous, displayed highly abnormal commissural axon structure of the central nervous system, and 70% of the flies homozygous for the mutant allele died prior to adulthood. Using high density SNP genotyping arrays, it was found that a mutation in PRKRA （the PACT/RAX gene） is the causative genetic mutation in DYT16, a novel autosomal recessive dystonia-parkinsonism syndrome in Brazilian patients.     

Activation of double-stranded RNA-activated protein kinase by mild impairment of oxidative metabolism in neurons

Thiamine (vitamin B1) deficiency (TD) causes mild and chronic impairment of oxidative metabolism and induces neuronal death in specific brain regions. The mechanisms underlying TD-induced cell death, however, remain unclear. The double-stranded RNA-activated protein kinase (PKR), has been well known for its anti-viral function. Upon activation by viral infection or double-stranded RNA, PKR phosphorylates its substrate, the α-subunit of eukaryotic initiation factor-2 (eIF2α), leading to inhibition of translation. In response to various cellular stresses, PKR can also be stimulated by its protein activators, or its mouse homologue, PKR activator (RAX). We demonstrated that TD in mice induced phosphorylation of PKR at Thr446 and Thr451 and phosphorylation of eIF2α at Ser51 in the cerebellum and the thalamus. TD caused phosphorylation of PKR and eIF2α, as well as nuclear translocation of PKR in primary cultures of cerebellar granule neurons. PKR phosphorylation is necessary for its nuclear translocation because TD failed to induce nuclear translocation of a T446A/T451A PKR mutant. Both PKR inhibitor and dominant-negative PKR mutant protected cerebellar granule neurons against TD-induced cell death. TD promoted the association between RAX and PKR. Antioxidant vitamin E dramatically decreased the RAX/PKR association and ameliorated TD-induced cell death. Our results indicate that TD-induced neuronal death is at least partially mediated by the activation of PKR.     

Lithium Fails to Protect Dopaminergic Neurons in the 6-OHDA Model of Parkinson’s Disease

Lithium has been used for the treatment of bipolar mood disorder and is shown to have neuroprotective properties. Since lithium inhibits the activity of glycogen synthase kinase 3 (GSK3) which is implicated in various human diseases, particularly neurodegenerative diseases, the therapeutic potential of lithium receives great attention. Parkinson’s disease (PD) is the second most common neurodegenerative disease, characterized by the pathological loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Intranigral injection of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) causes selective and progressive degeneration of dopaminergic neurons in SNpc, and is a commonly used animal model of PD. The current study was designated to determine whether lithium is effective in alleviating 6-OHDA-induced neurodegeneration in the SNpc of rats. We demonstrated that chronic subcutaneous administration of lithium inhibited GSK3 activity in the SNpc, which was evident by an increase in phosphorylation of GSK3β at serine 9, cyclin D1 expression, and a decrease in tau phosphorylation. 6-OHDA did not affect GSK3 activity in the SNpc. Moreover, lithium was unable to alleviate 6-OHDA-induced degeneration of SNpc dopaminergic neurons. The results suggest that GSK3 is minimally involved in the neurodegeneration in the rat 6-OHDA model of PD.