A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.     

Targeted Depletion of TDP-43 Expression in the Spinal Cord Motor Neurons Leads to the Development of Amyotrophic Lateral Sclerosis-like Phenotypes in Mice

ALS, or amyotrophic lateral sclerosis, is a progressive and fatal motor neuron disease with no effective medicine. Importantly, the majority of the ALS cases are with TDP-43 proteinopathies characterized with TDP-43-positive, ubiquitin-positive inclusions (UBIs) in the cytosol. However, the role of the mismetabolism of TDP-43 in the pathogenesis of ALS with TDP-43 proteinopathies is unclear. Using the conditional mouse gene targeting approach, we show that mice with inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbp(lx/-)) exhibit progressive and male-dominant development of ALS-related phenotypes including kyphosis, motor dysfunctions, muscle weakness/atrophy, motor neuron loss, and astrocytosis in the spinal cord. Significantly, ubiquitinated proteins accumulate in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre-Tardbp(lx/-) mice with the ALS phenotypes. This study not only establishes an important role of TDP-43 in the long term survival and functioning of the mammalian spinal cord motor neurons, but also establishes that loss of TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.     

人野生型p53基因增强5-FU对大肠癌化疗敏感性的分子生物学机制

为了探讨人野生型p53（wt-p53）基因增强大肠癌细胞化疗敏感性的分子生物学机制,将携带wt p53基因的质粒分别转染两种p53基因突变的人大肠癌细胞系HT-29及SW620,分析细胞中p53及细胞周期蛋白D1（cyclin D1）蛋白的表达水平;将化疗药物5 氟尿嘧啶（5-fluorouracil,5-FU）以不同浓度、不同时间分别作用于HT-29及SW620细胞,另外将已转染wt-p53基因的大肠癌细胞用5-FU进行诱导,Western印迹分析上述干预条件下细胞中p53蛋白及细胞周期蛋白D1表达水平的变化;流式细胞术检测wt p53基因联合5-FU组及对照组中细胞凋亡的改变情况.结果表明,wt-p53基因能增加癌细胞中细胞周期蛋白D1的表达,与wt-p53基因呈剂量依赖性关系;5-FU则降低其蛋白表达,与5-FU呈时间和剂量依赖性关系,而5-FU所致的细胞周期蛋白D1表达水平的降低在细胞预先转染了wt- p53基因时会被抑制;wt-p53基因与5-FU联合使用能提高大肠癌细胞凋亡率.结果提示,wt-p53基因可提高大肠癌细胞中细胞周期蛋白D1的表达水平,并抑制5-FU所致的细胞周期蛋白D1降解,从而提高大肠癌细胞对化疗药物5-FU的敏感性.     

Mice Carrying ALS Mutant TDP-43, but Not Mutant FUS,Display In Vivo Defects in Axonal Transport of Signaling Endosomes

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Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

Reactivation of human cytomegalovirus (HCMV) can cause severe disease in recipients of hematopoietic stem cell transplantation. Although preclinical research in murine models as well as clinical trials have provided ''proof of concept'' for infection control by pre-emptive CD8 T-cell immunotherapy, there exists no predictive model to experimentally evaluate parameters that determine antiviral efficacy of human T cells in terms of virus control in functional organs, prevention of organ disease, and host survival benefit. We here introduce a novel mouse model for testing HCMV epitope-specific human T cells. The HCMV UL83/pp65-derived NLV-peptide was presented by transgenic HLA-A2.1 in the context of a lethal infection of NOD/SCID/IL-2rg^-/- mice with a chimeric murine CMV, mCMV-NLV. Scenarios of HCMV-seropositive and -seronegative human T-cell donors were modeled by testing peptide-restimulated and T-cell receptor-transduced human T cells, respectively. Upon transfer, the T cells infiltrated host tissues in an epitope-specific manner, confining the infection to nodular inflammatory foci. This resulted in a significant reduction of viral load, diminished organ pathology, and prolonged survival. The model has thus proven its potential for a preclinical testing of the protective antiviral efficacy of HCMV epitope-specific human T cells in the evaluation of new approaches to an immunotherapy of CMV disease.     

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.     

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: Characterization of inhibition by nucleic acids and 4-aminoquinolines

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261–414 aa) with K_D values of 6.2 nM and 8.7 nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170–414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.     

PEMT基因V175M多态性与非酒精性脂肪肝易感性的关系

目的:探讨磷脂酰乙醇胺N-甲基转移酶(phosphatidylethanolamine N-methyltransferase,PEMT)基因V175M多态性与非酒精性脂肪肝(NAFLD)易感性的关系。方法:抽取NAFLD患者外周血基因组DNA,PCR扩增获得其V175M基因,测序后分析PEMT基因V175M多态位点的基因分型。收集患者身高、体重、腰围、空腹血糖、低密度脂蛋白、空腹胰岛素等临床资料,计算体重指数和HOMA胰岛素抵抗指数(HOMA-IR),同时选取年龄、性别相匹配的非NAFLD79人为对照。结果:NAFLD组和对照组在年龄、性别方面相匹配,但NAFLD组的肥胖者显著增多。NAFLD组和对照组M等位基因的频率分别为20.1%和12.0%,差异有统计学意义(P<0.05),NAFLD组和对照组VM MM基因型的频率分别为26.6%和22.8%(P=0.05)。以BMI≥25作为肥胖的标准,将研究对象分为肥胖和非肥胖者,在肥胖的对照和NAFLD组中,V175M等位基因和基因型频率差异均无统计学意义(P>0.05);在非肥胖的NAFLD和对照组中,M等位基因的频率分别为21.9%和9.6%,VM基因型的频率则分别为43.8%及19.3 %,差异均有统计学意义(P<0.05)。通过引入其他已知的非肥胖型NAFLD易感因素(腰围,LDL和HOMA-IR),再进行Logistic回归分析,V175M基因型未能进入回归方程,P=0.323。结论:广州汉族人中存在PEMT V175M多态性,且以V等位基因为主,PEMT V175M并非NAFLD的独立易感因素。     

Soybean-derived phosphatidylinositol inhibits in vivo low concentrations of amyloid beta protein-induced degeneration of hippocampal neurons in V337M human tau-expressing mice

In our previous reports using primary cultured rat hippocampal neurons, pathophysiological concentrations (< or =10 nM) of amyloid beta proteins (Abetas) showed neurotoxicity via a phosphatidylinositol metabolism disorder, and soybean-derived phosphatidylinositol protected the neurons against the Abeta's neurotoxicity. In the present study, such a neurotoxic effect of Abeta and a neuroprotective effect of phosphatidylinositol were examined in vivo using transgenic mice expressing V337 M human tau. Intrahippocampal CA1 injection of 1.5 mul of 100 nM or 1 microM Abeta25-35 increased the number of degenerating neurons with an apoptotic feature in bilateral hippocampal CA1, CA2, CA3 and dentate gyrus regions in 1 month, demonstrating an in vivo neurotoxic effect of Abeta at lower concentrations after diffusion. Intrahippocampal co-injection or intracerebroventricular administration of 1.5 microl of 500 nM phosphatidylinositol prevented the Abeta25-35-induced neuronal degeneration in all the hippocampal regions, while co-injection of another acidic phospholipid, phosphatidylserine (1.5 microl, 500 nM) with Abeta25-35 showed no protective effects. Thus, exogenously applied phosphatidylinositol appeared to minimize the toxic effects of Abeta in vivo. These results suggest that soybean-derived phosphatidylinositol may be effective in the treatment of Alzheimer's disease.     

Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication.

The DNA from a wide variety of human tumors has sustained mutations within the conserved p53 coding regions. We have purified wild-type and tumor-derived mutant p53 proteins expressed from baculovirus vectors and examined their interactions with SV40 DNA. Using DNAase I footprinting assays, we observed that both human and murine wild-type p53 proteins bind specifically to sequences adjacent to the late border of the viral replication origin. By contrast, mutant p53 proteins failed to bind specifically to these sequences. SV40 T antigen prevented wild-type p53 from interacting with this region. These data show that normal but not oncogenic forms of p53 are capable of sequence-specific interactions with viral DNA. Furthermore, they provide insights into the mechanisms by which viral proteins might regulate the control of viral growth and cell division.     

Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly

The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. We previously characterized a new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. In the present study, we demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub⁺) TBPH pathology. Although we found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, we herein showed that an augmented soluble cytoplasmic Ub⁺-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. We demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend our knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases.     

Increased Oxidative Damage in Carriers of the Germline TP53 p.R337H Mutation

Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H). The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17) and non-carriers (NC, n = 17). We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively). Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.20±0.71, C = 160.5±0.88, P<0.0001). Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis.