Expression and Cellular Distribution of Vascular Endothelial Growth Factor‐C System in Cortical Tubers of the Tuberous Sclerosis Complex

Cortical tubers are malformations of cortical development in patients with tuberous sclerosis complex (TSC), and highly associated with pediatric intractable epilepsy. Recent evidence has shown that signaling mediated through vascular endothelial growth factor‐C (VEGF‐C) and its receptors, VEGFR‐2 and VEGFR‐3, has direct effects on both neurons and glial cells. To understand the potential role of VEGF‐C system in the pathogenesis of cortical tubers, we investigated the expression patterns of VEGF‐C signaling in cortical tubers compared with age‐matched normal control cortex (CTX). We found that VEGF‐C, VEGFR‐2 and VEGFR‐3 were clearly upregulated in tubers at both the mRNA and protein levels, compared with CTX. The in situ hybridization and immunostaining results demonstrated that VEGF‐C, VEGFR‐2 and VEGFR‐3 were highly expressed in dysplastic neurons (DNs), giant cells (GCs) and reactive astrocytes within tubers. Most DNs/GCs expressing VEGF‐C and its receptors co‐labeled with neuronal rather than astrocytic markers, suggesting a neuronal lineage. In addition, protein levels of Akt‐1, p‐Bad and ERK1/2, the important downstream factors of the VEGF‐C pathway, were significantly increased in cortical tubers, indicating involvement of VEGF‐C–dependent prosurvival signaling in cortical tubers. Taken together, our results suggest a putative role for the VEGF‐C signaling pathway in the pathogenesis of cortical tubers.     

Functional Assessment of TSC2 Variants Identified in Individuals with Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Here, we investigate the effects of 78 TSC2 variants identified in individuals suspected of TSC, on the function of the TSC1–TSC2 complex. According to our functional assessment, 40 variants disrupted the TSC1–TSC2‐dependent inhibition of TORC1. We classified 34 of these as pathogenic, three as probably pathogenic and three as possibly pathogenic. In one case, a likely effect on splicing as well as an effect on function was noted. In 15 cases, our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our data support the notion that different, nonterminating TSC2 mutations can have distinct effects on TSC1–TSC2 function, and therefore, on TSC pathology.     

Distinct clinical characteristics of Tuberous Sclerosis Complex patients with no mutation identified

Tuberous Sclerosis Complex (TSC) is a multi-system disorder that is highly variable in its clinical presentation. Current molecular diagnostic methods permit identification of mutations in either TSC1 or TSC2 in 75–85% of TSC patients. Here we examine the clinical characteristics of those TSC patients who have no mutation identified (NMI). A retrospective review of our patient population that had comprehensive testing for mutations in TSC1 / TSC2 identified 23/157 (15%) that were NMI. NMI patients had a lower incidence of brain findings on imaging studies, neurological features, and renal findings than those with TSC2 mutations. In contrast, NMI patients had a lower incidence of seizures than TSC patients with TSC1 mutations, but had a higher incidence of both renal angiomyolipomas and pulmonary lymphangioleiomyomatosis. This distinct constellation of findings suggest that NMI patients may have a unique molecular pathogenesis, different from that seen in TSC patients with the usual mutations in TSC1 and TSC2 . We suggest that the mechanisms of disease in these patients include both mosaicism for a TSC2 mutation, and unusual non-coding region mutations in TSC2 .     

Healthcare transition from childhood to adulthood in Tuberous Sclerosis Complex

Healthcare transition from childhood to adulthood is required to ensure continuity of care of an increasing number of individuals with chronic conditions surviving into adulthood. The transition for patients with tuberous sclerosis complex (TSC) is complicated by the multisystemic nature of this condition, age-dependent manifestations, and high clinical variability and by the presence of intellectual disability in at least half of the individuals. In this article, we address the medical needs regarding each TSC-related manifestation in adulthood, and the services and support required. We review existing models of transition in different chronic conditions, discuss our experience in transitioning from the pediatric to the adult TSC Clinic at our Institution, and propose general rules to follow when establishing a transition program for TSC. Although a generalizable transition model for TSC is likely not feasible for all Institutions, a multidisciplinary TSC clinic is probably the best model, developed in accordance with the resources available and country-specific healthcare systems. Coordination of care and education of the adult team should be always sought regardless of the transition model.     

Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex

The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.     

Central Nervous System Pathology of Tuberous Sclerosis in Children

Ten cases of tuberous sclerosis involving the central nervous system (CNS) in children aged 2 days to 15 years were studied. The abnormal cells found in subependymal, cortical, and white matter lesions were examined by light and electron microscopy. Histochemistry and immunohistochemistry were also employed. The results were similar in all lesions. Approximately one-third of the abnormal cells were positive by glial fibrillary acidic protein (GFAP), one-half by Nissl, and one-quarter by Holzer's stains. The intensity and pattern of GFAP staining varied from cell to cell and could not be predicted before staining.

Ultrastructurally the cytoplasm of abnormal cells contained moderate to large numbers of 9-to 12-nm diameter fibrils and frequent dense bodies with crystalline appearance. Stacked rough endoplasmic reticulum was variable. Cell junctions and glycogen were rare. Nuclei were usually vesicular with a prominent nucleolus.

Individual cells of tuberous sclerosis have features of both neurons and astrocytes. The disease may affect cells before differentiation. The predominant abnormal features of the cells in tuberous sclerosis are a great increase in fibrils and the presence of dense bodies, which may be a nonspecific reaction or result from a metabolic defect affecting the cells.     

Central Nervous System Pathology of Tuberous Sclerosis in Children

Ten cases of tuberous sclerosis involving the central nervous system (CNS) in children aged 2 days to 15 years were studied. The abnormal cells found in subependymal, cortical, and white matter lesions were examined by light and electron microscopy. Histochemistry and immunohistochemistry were also employed. The results were similar in all lesions. Approximately one-third of the abnormal cells were positive by glial fibrillary acidic protein (GFAP), one-half by Nissl, and one-quarter by Holzer's stains. The intensity and pattern of GFAP staining varied from cell to cell and could not be predicted before staining.

Ultrastructurally the cytoplasm of abnormal cells contained moderate to large numbers of 9-to 12-nm diameter fibrils and frequent dense bodies with crystalline appearance. Stacked rough endoplasmic reticulum was variable. Cell junctions and glycogen were rare. Nuclei were usually vesicular with a prominent nucleolus.

Individual cells of tuberous sclerosis have features of both neurons and astrocytes. The disease may affect cells before differentiation. The predominant abnormal features of the cells in tuberous sclerosis are a great increase in fibrils and the presence of dense bodies, which may be a nonspecific reaction or result from a metabolic defect affecting the cells.     

结节性硬化症基因型与表型研究进展

结节性硬化症是一种以全身多种组织错构瘤样增生为主要特征的常染色体显性遗传性疾病,具有遗传异质性,TSC1和TSC2为其致病基因。近年来对结节性硬化症基因型与表型的广泛研究,为该病预防和临床诊治提供了一定的依据。     

人LC3蛋白在大肠杆菌中的表达纯化及其抗体的制备与鉴定

目的:制备兔抗人LC3的多克隆抗体,为细胞自噬的研究提供有用的检测工具。方法:构建pET32a(+)LC3的原核细胞表达载体并进行序列鉴定,大肠杆菌BL21(DE3)中诱导表达LC3蛋白,SP-Sepharose XL柱纯化;纯化的人LC3蛋白免疫兔制备抗血清,通过LC3蛋白偶联的Sepharose 4B(LC3-Sepharose 4B)亲和层析纯化兔抗人LC3的多克隆抗体,以间接ELISA和Western blot鉴定其特异性。结果:成功实现了LC3蛋白在大肠杆菌中的高效表达和纯化,并用质谱分析证明表达蛋白相对分子质量(Mr)为15 500。制备的兔抗人LC3多克隆抗体具有很高的特异性,与大肠杆菌成分无交叉反应。Western blot能够特异检测自噬过程中LC3Ⅰ型和Ⅱ型蛋白,观察到自噬发生时LC3Ⅰ向LC3Ⅱ的转化。结论:获得了重组人LC3蛋白,成功地制备了该分子的特异性多克隆抗体,为细胞自噬的检测提供了有用的探针。     

Genotype/phenotype correlation in 123 Chinese patients with Tuberous Sclerosis Complex

Tuberous Sclerosis Complex (TSC) is a multisystemic neurocutaneous disorder with autosomal dominant inheritance. We performed mutation analyses on 123 Chinese patients with “definite TSC” according to the latest diagnostic criteria. Pathogenic / likely-pathogenic variants were identified in 72.2% of all index patients (70/97), in which 35.7% (25/70) had TSC1 variants and 64.3% (45/70) had TSC2 variants. 84.5% (82/97) cases were sporadic and 15.5% (15/97) cases were familial. 62 unique variants were reported, in which 41.9% (26/62) were novel. Male patients had significantly more subependymal nodules (p=0.029) than females, whereas renal angiomyolipoma (p=0.032) occurred predominantly in females. Sporadic cases also had more renal angiomyolipoma (p=0.004), cortical tubers (p=0.008), hypopigmented macules (p=0.018) and fibrous cephalic plaques (p=0.028) than cases with known inheritance. Patients with TSC2 pathogenic variants were more likely to have mental retardation (p<0.001), cardiac rhabdomyoma (p=0.004), renal angiomyolipoma (p=0.006) and facial angiofibromas (p=0.026) than those with TSC1 pathogenic variants, while mutation-negative cases showed a mixed phenotype between those with TSC1 and TSC2 variants. There were no significant phenotypic differences between patients with and without TSC1/TSC2 variants, but TSC2 missense and in-frame variants were associated with higher frequencies of mental retardation (P<0.001), renal angiomyolipoma (p=0.001), cardiac rhabdomyoma (p=0.012) and facial angiofibroma (p=0.021) than those with TSC1 frameshift and splice site variants. Furthermore, a higher frequency of mental retardation (p=0.013) was observed in patients with TSC2 missense and in-frame variants than those with frameshift and splice site variants. All 14 antenatal-onset patients had cardiac rhabdomyoma. They had fewer seizures (p=0.028) than patients with paediatric-onset, but were more likely to have mental retardation (p=0.035) than individuals with adult-onset disease. Generally, paediatric-onset patients had more neurological manifestations, while initial presentations of adult-onset TSC were more diverse.     

Crystalline Inclusions in a Subependymal Giant Cell Tumor in a Patient with Tuberous Sclerosis

Unusual crystalline cytoplasmic inclusions were encountered in tumor cells in a subependymal giant cell tumor (SEGT) in a 16-year-old girl with tuberous sclerosis. By electron microscopy, the tumor cells demonstrated typical features previously described in SEGT, including abundant dense bodies, prominent Golgi complexes, abundant mitochondria, rough and smooth endoplasmic reticulum, scattered intermediate filaments and microtubules, glycogen, and rare synaptic contacts as well as primitive intercellular junctions. The dense bodies were bound by a single membrane and were round, ovoid, irregular, or cylindric in appearance with electron-dense homogenous content or fingerprint profiles. Of note was the presence of numerous cytoplasmic rhomboidal or rectangular crystalline inclusions akin to those seen in alveolar soft part sarcoma. These inclusions measured as much as 8 μm in length and had 7-nm periodicities, often with intersecting lamellae. Rarely, the membrane-bound dense bodies showed areas of similar periodicities, indicating that the crystalline inclusions are related to and might originate from the dense bodies. While crystalline inclusions have previously been described in one patient with SEGT (Bender and Yunis, Ultrastruct Pathol 1980; 1:287-299), the inclusions in the present case were a striking feature and add to the spectrum of the ultrastructural pathology of SEGT.     

子宫颈鳞状细胞癌组织中LC3B的表达及其与Ki-67表达的相关性

目的：探讨自噬标志物LC3B蛋白在子宫颈鳞状细胞癌组织中的表达及其与细胞增殖蛋白Ki-67表达的相关性。方法采用免疫组化SP法检测16例正常子宫颈和126例子宫颈鳞状细胞癌组织中LC3B及Ki-67的表达。结果 LC3B蛋白在子宫颈鳞状细胞癌组织中的表达低于正常子宫颈上皮,差异有统计学意义(P<0．05);LC3B与 Ki-67表达呈负相关(rs =-0．248,P<0．05)。结论子宫颈鳞状细胞癌中LC3B的表达降低,自噬活性减弱可能促进早期子宫颈鳞状细胞癌细胞的增殖,该发现为进一步探究自噬在子宫颈癌发生、发展中的作用提供临床参考依据。     

乳腺癌中自噬相关基因LC3与凋亡相关基因XIAP的表达及意义

目的观察自噬相关基因LC3和凋亡相关基因XIAP在乳腺癌组织中的表达,探讨其表达与乳腺癌临床病理特征的关系。方法采用组织芯片平台,应用免疫组化法检测121例乳腺癌组织中LC3、XIAP的表达,并结合临床病理因素进行分析。结果乳腺癌组织中,LC3和XIAP的高表达率分别为84.2%(102/121)和90.9%(110/121)。LC3的高表达与HER-2过表达呈显著正相关(P0.05),XIAP的高表达与Ki-67和HER-2过表达呈显著正相关(P均0.05),但均与患者年龄、肿瘤大小、组织学分级、TNM分期、淋巴结转移、ER和PR表达无关(P均0.05)。经Spearman秩相关检验,LC3和XIAP在乳腺癌中的表达呈显著正相关(P0.05)。结论在乳腺癌中,高自噬活性与低凋亡现象并存,HER-2可能参与其中。     

Developmental expression of LC3alpha and beta: absence of fibronectin or autophagy phenotype in LC3beta knockout mice.

Murine light chain 3 (LC3) exists as two isoforms, LC3alpha and beta: LC3beta is an RNA-binding protein that enhances fibronectin (FN) mRNA translation, and is also a marker of autophagy. We report embryonic expression patterns for LC3alpha and LC3beta, with some overlap but notable differences in the brain, and in tissues of non-neuronal origin. LC3beta knockout (-/-) mice develop normally without a compensatory increase in LC3alpha. LC3beta-/- embryonic fibroblasts (MEFs) exhibit reduced FN synthesis but maintain wild type (WT) levels of FN protein. No significant changes in proteins associated with FN turnover, i.e., caveolin-1, LRP-1, or matrix metalloproteinases were identified. Autophagosomes form in amino acid-starved LC3beta-/-MEFs, and Caesarean-delivered pups survive as long as WT pups without an increase in LC3-related proteins linked to autophagy. These results suggest novel compensatory mechanisms for loss of LC3beta, ensuring proper FN accumulation and autophagy during fetal and neonatal life.     

人肝细胞癌中LC3B和p62的表达及意义

目的观察自噬标志因子LC3B和p62在人肝细胞癌(hepatocellular carcinoma,HCC)组织中的表达及其临床意义。方法采用免疫组化法检测61例人HCC组织中LC3B和p62的表达,分析二者表达与临床病理特征及其预后的关系。结果 HCC中LC3B和p62的高表达率分别为31.1%和65.6%;LC3B表达与肿瘤大小、组织学分级和临床分期呈显著正相关(P0.05),p62表达与肿瘤临床分期呈显著正相关(P0.05);LC3B和p62表达均与HCC患者的无复发生存期和总生存期密切相关(P=0.009和0.036;P=0.001和0.007)。结论肿瘤细胞自噬活性的提高可能参与人HCC的发展,LC3B和p62表达可能是肝细胞进展和预后不良的潜在标志物。     

地塞米松诱导的危重病性肌病大鼠骨骼肌Beclin1、LC3的表达

目的 　观察地塞米松对大鼠危重病性肌病（CIM）比目鱼肌细胞自噬相关因子Beclin1和LC3表达的影响,探讨地塞米松诱导的大鼠危重病性肌病的可能机制。 方法　将健康SD大鼠分为对照组和实验组;实验组又分为7,9,11d 3个时相点（n=10）。实验组采用5mg/kg地塞米松连续腹腔注射,每天1次,对照组腹腔注射等量的生理盐水。采用足迹法判定肌功能缺损情况。利用免疫组化和Western blot检测比目鱼肌细胞自噬相关因子Beclin1和LC3的表达情况。 结果　与对照组相比,实验组大鼠出现不同程度肌肉功能缺损症状,以11d时大鼠缺损程度最为严重。Western blot结果显示,对照组或可见微弱的Beclin1、LC3阳性表达,随着时间延长,实验组可见明显的Beclin1、LC3阳性表达,以11d时相点表达最为明显。免疫组化结果也证实了同一趋势。 结论　地塞米松诱导的大鼠CIM可能通过激活Beclin1和LC3的表达从而发挥对细胞自噬的调节作用。     

Autoantibodies against the autophagic protein microtubule-associated light-chain 3 (LC3): Immunocharacterization of an atypical ANA pattern

Abstract

Autoantibodies to nuclear and cytoplasmic antigens are commonly detected by indirect immunofluorescence (IIF) on HEp-2 cells, and three major staining patterns (nuclear, cytoplasmic, and mitotic) are distinguished. Here, we report an atypical cytoplasmic pattern, not described so far, observed in the serum of a patient with a controversial diagnosis of systemic lupus erythematosus (SLE). Moreover, for the first time, we have revealed the presence of autoantibodies against the microtubule-associated light-chain 3 (LC3) protein, which plays a key role in the autophagic process. The target antigen has been identified in IIF by means of a competition test using purified anti-LC3 antibodies on HEp-2 cells, and confirmed by Western blot analysis using cellular or recombinant LC3 as antigen, immunoreacted with the patient’s serum. The identification of this atypical pattern and the related autoantibody-antigen system sheds new light on autophagy, which is increasingly considered to be involved in the etiopathogenesis of autoimmune disorders, and could contribute to select more personalized therapies.     

大肠癌中Beclin1、LC3和mTOR的表达及意义

目的：探讨Beclin1、LC3和mTOR在大肠癌中的表达及意义。方法采用免疫组化EnVision法、Western blot和RT-PCR技术检测Beclin1、LC3和mTOR在大肠癌中的表达,并结合临床病理因素进行分析。结果 Beclin1、LC3和mTOR在大肠癌中的阳性率分别为90．50%、87．19%和46．28%,均明显高于癌旁组织( P<0．05),且LC3蛋白在中+低分化大肠癌中的表达(91．34%、89．58%)高于高分化大肠癌(77．61%),在有淋巴结转移组的表达(95．41%)高于无淋巴结转移组(80．45%), mTOR在有淋巴结转移组的表达(57．80%)高于无淋巴结转移组(35．33%)(P<0．05),Beclin1过表达与大肠癌分化程度和淋巴结转移均无关(P>0．05)。 LC3与Beclin1在大肠癌中表达呈正相关(rs =0．593,P<0．01),与mTOR表达呈负相关(rs =-0．165,P<0．01),Beclin1和mTOR表达之间无明显相关性(P>0．05)。 Kaplan-Meier生存分析显示,Beclin1和LC3均阳性、mTOR阴性、无淋巴结转移的大肠癌患者5年生存率高于Beclin1和LC3均阴性、mTOR阳性及有淋巴结转移的患者。多因素分析显示,LC3、mTOR和淋巴结转移是影响大肠癌预后的独立因素。 RT-PCR和Western blot检测结果显示：Beclin1、LC3和mTOR mRNA和蛋白表达水平均明显高于癌旁组织(P<0．05)。结论自噬相关基因Beclin1、LC3和mTOR的异常表达可能与大肠癌的发生、发展及预后相关;联合检测Beclin1、LC3和mTOR在大肠癌中的表达有助于评估进展程度和预后判断。