Effects of Small Molecules on Chaperone-Mediated Autophagy

Autophagy, including macroautophagy (MA), chaperone-mediated autophagy (CMA), crinophagy, pexophagy and microautophagy, are processes by which cells select internal components such as proteins, secretory vesicles, organelles, or foreign bodies, and deliver them to lysosomes for degradation. MA and CMA are activated during conditions of serum withdrawal in cell culture and during short-term (MA) and prolonged (CMA) starvation in organisms. Although MA and CMA are activated under similar conditions, they are regulated by different mechanisms. We used pulse/chase analysis under conditions in which most intracellular proteolysis is due to CMA to test a variety of compounds for effects on CMA. We show that inhibitors of MA such as 3-methyladenine, wortmannin, and LY294002 have no effect on CMA. Protein degradation by MA is sensitive to microtubule inhibitors such as colcemide and vinblastine, but protein degradation by CMA is not. Activators of MA such as rapamycin also have no effect on CMA. We demonstrate that CMA, like MA, is inhibited by protein synthesis inhibitors anisomycin and cycloheximide. CMA is also partially inhibited when the P38 mitogen activated protein kinase is blocked. Finally we demonstrate that the glucose-6-phophate dehydrogenase inhibitor, 6-aminonicotinamide, and heat shock protein of 90 kilodaltons inhibitor, geldanamycin, have the ability to activate CMA.     

神经细胞粘附分子与记忆

记忆的形成阶段包含着神经元突触的可塑性变化过程.近年来的研究表明,神经细胞粘附分子可同时增进突触的可塑性和维持突触结构的稳定性.许多研究证实神经细胞粘附分子对与学习和记忆相关的过程起着一定的调节作用.     

Small Molecules Facilitate the Reprogramming of Mouse Fibroblasts into Pancreatic Lineages

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Melatonin is Involved in Regulation of the Expression of Neural Cell Adhesion Molecules in the Rat Brain

Cell adhesion molecules play a diverse role in neural development, signal transduction, structural linkage to extracellular and intracellular proteins, synaptic stabilization, neurogenesis, and learning. Neural cell adhesion molecules (NCAM) are members of the immunoglobulin superfamily and are involved in synaptic rearrangements in the mature brain. There are three major NCAM isoforms: NCAM 180, NCAM 140, and NCAM 120. Several studies reported that NCAM play a central role in memory formation. We investigated the effects of melatonin on the expression of NCAM in the hippocampus, cortex, and cerebellum of rats. The levels of NCAM isoforms were determined by Western blotting. After administration of melatonin for 7 days, the expression of NCAM 180 increased both in the hippocampus and in the cortex, as compared with the control. In contrast, in rats exposed to constant illumination for 7 days (a procedure that inhibits endogenous production of melatonin), levels of NCAM 180 dropped in the hippocampus and became undetectable in the cortex and cerebellum. Levels of NCAM 140 in the hippocampus of light-exposed rats also decreased. There was no change in the expression of NCAM 120 in any brain region. This is the first report indicating that melatonin exerts a modulatory effect on the expression of NCAM in brain areas related to realization of cognitive functions. Melatonin may be involved in structural remodeling of synaptic connections during memory and learning processes.     

The PEA-15 Protein Regulates Autophagy via Activation of JNK

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.     

Foxp1 Regulates Neural Stem Cell Self-Renewal and Bias Toward Deep Layer Cortical Fates

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Small Molecules Enable Cardiac Reprogramming of Mouse Fibroblasts with a Single Factor,Oct4

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New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS.     

Bilirubin Inhibits Tumor Cell Growth via Activation of ERK

Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.     

Developmental Expression of Neural Cell Adhesion Molecules of Oligodendrocytes In Vivo and in Culture

Abstract: Previously, we have shown that oligodendrocyte adhesion molecules are related to the 120,000–M_r neural cell adhesion molecule (NCAM-120). In this report, we present further evidence that the oligodendrocyte adhesion molecule is NCAM-120. Studies on the expression of NCAM-120 and other molecular forms of NCAM in vivo in rat brain, in vitro in primary mixed cultures, and in cultures enriched for oligodendrocytes are described. Western blot analysis of rat brain using anti-NCAM showed that NCAM-120 first appears at postnatal day 7 and increases in quantity thereafter, coincident with the development of oligodendrocytes in vivo and comparable to the expression of myelin basic protein. Purified oligodendrocytes from 4-week-old rat brains expressed only NCAM-120. Quantitation of various forms of NCAMs in rat brain showed marked age-related differences in the expression of three molecular forms of NCAM. Immunofluorescence analysis showed that oligodendrocytes, at all ages tested, expressed NCAM, but in older oligodendrocytes, the intensity of staining was less. Western blot analysis of oligodendrocyte-enriched cultures showed that from day 1 after isolation (12 days of age) through day 7 after isolation (18 days of age) only NCAM-120 is seen. A possible role for NCAM in myelination and remyelination is discussed.     

INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal,Reprogramming, and Blastocyst Development

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细胞自噬在吉非替尼治疗非小细胞肺癌中的作用及其机制研究

目的:观察吉非替尼对非小细胞肺癌细胞自噬的影响,并探讨其机制。方法:将BalB/CA-nu品系裸小鼠分成两组,各组20只,均造肺癌模型,其中吉非替尼治疗组小鼠在肺癌组模型的基础上给予吉非替尼25mg/kg 14d,停药后剖杀。取组织切片,通过免疫荧光、RT-PCR以及Western blot的试验方法检测自噬相关基因Beclin1和MAPLC3的表达。结果:免疫荧光、RT-PCR以及Western blot的试验方法检测均发现肺癌组小鼠组织中Beclin1和MAPLC3表达较低,而在吉非替尼处理组中,Beclin1和MAPLC3的表达明显升高,差异有统计学意义。结论:吉非替尼可以通过增强细胞自噬从而发挥对非小细胞肺癌的抑制作用。     

ERM-Dependent Assembly of T Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli prior to Activation

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A Phylogenetic Analysis of the L1 Family of Neural Cell Adhesion Molecules

L1-type genes form one of several distinct gene families that encode adhesive proteins, which are predominantly expressed in developing and mature metazoan nervous systems. These proteins have a multitude of different important cellular functions in neuronal and glial cells. L1-type gene products are transmembrane proteins with a characteristic extracellular domain structure consisting of six immunoglobulin and three to five fibronectin type III protein folds. As reported here, L1-type proteins can be identified in most metazoan phyla with the notable exception of Porifera (sponges). This puts the origin of L1-type genes at a point in time when primitive cellular neural networks emerged, approximately 1,200 to 1,500 million years ago. Subsequently, several independent gene duplication events generated multiple paralogous L1-type genes in some phyla, allowing for a considerable diversification of L1 structures and the emergence of new functional features and molecular interactions. One such evolutionary newer feature is the appearance of RGD integrin-binding motifs in some vertebrate L1 family members.     

Derivation and Expansion Using Only Small Molecules of Human Neural Progenitors for Neurodegenerative Disease Modeling

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.     

At the Crossroads of Chemistry and Cell Biology: Inhibiting Membrane Traffic by Small Molecules

Intracellular membrane traffic regulates cell physiology at multiple levels ranging from cell growth and development to the function of the nervous and immune systems. Multiple endocytic routes are used by distinct cargoes including ligands bound to their receptors but also viruses and pathogens to gain access to the cell interior. Within the endosomal system, proteins and lipids are sorted for degradation or recycling allowing cells to dynamically respond to environmental signals and to regulate cell shape and morphology. Some receptors or toxins are sorted along the retrograde pathway from endosomes to the Golgi complex, where they intersect with secretory cargo destined for exocytosis. Genetic manipulations of these pathways frequently cause problems with regard to data interpretation as the resulting phenotypes may be indirect consequences resulting from perturbation of multiple steps or trafficking routes. Hence, novel approaches are needed to acutely and reversibly perturb intracellular membrane traffic, e.g. by small molecule inhibitors. Such drugs may also be pharmacologically important as they offer new avenues to fight human diseases. Here, we provide an overview of the small molecules available to interfere with intracellular membrane traffic and outline strategies for future research.