Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice

Autophagy is an intracellular bulk degradation process through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Although the primary role of autophagy in many organisms is in adaptation to starvation, autophagy is also thought to be important for normal turnover of cytoplasmic contents, particularly in quiescent cells such as neurons. Autophagy may have a protective role against the development of a number of neurodegenerative diseases. Here we report that loss of autophagy causes neurodegeneration even in the absence of any disease-associated mutant proteins. Mice deficient for Atg5 (autophagy-related 5) specifically in neural cells develop progressive deficits in motor function that are accompanied by the accumulation of cytoplasmic inclusion bodies in neurons. In Atg5-/- cells, diffuse, abnormal intracellular proteins accumulate, and then form aggregates and inclusions. These results suggest that the continuous clearance of diffuse cytosolic proteins through basal autophagy is important for preventing the accumulation of abnormal proteins, which can disrupt neural function and ultimately lead to neurodegeneration.     

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.     

The role of autophagy during the early neonatal starvation period

At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3-12 h before returning to basal levels within 1-2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (approximately 12 h) is much shorter than that of wild-type mice (approximately 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of 'self' proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.     

HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS

A prominent feature of late-onset neurodegenerative diseases is accumulation of misfolded protein in vulnerable neurons. When levels of misfolded protein overwhelm degradative pathways, the result is cellular toxicity and neurodegeneration. Cellular mechanisms for degrading misfolded protein include the ubiquitin-proteasome system (UPS), the main non-lysosomal degradative pathway for ubiquitinated proteins, and autophagy, a lysosome-mediated degradative pathway. The UPS and autophagy have long been viewed as complementary degradation systems with no point of intersection. This view has been challenged by two observations suggesting an apparent interaction: impairment of the UPS induces autophagy in vitro, and conditional knockout of autophagy in the mouse brain leads to neurodegeneration with ubiquitin-positive pathology. It is not known whether autophagy is strictly a parallel degradation system, or whether it is a compensatory degradation system when the UPS is impaired; furthermore, if there is a compensatory interaction between these systems, the molecular link is not known. Here we show that autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, and that histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase that interacts with polyubiquitinated proteins, is an essential mechanistic link in this compensatory interaction. We found that compensatory autophagy was induced in response to mutations affecting the proteasome and in response to UPS impairment in a fly model of the neurodegenerative disease spinobulbar muscular atrophy. Autophagy compensated for impaired UPS function in an HDAC6-dependent manner. Furthermore, expression of HDAC6 was sufficient to rescue degeneration associated with UPS dysfunction in vivo in an autophagy-dependent manner. This study suggests that impairment of autophagy (for example, associated with ageing or genetic variation) might predispose to neurodegeneration. Morover, these findings suggest that it may be possible to intervene in neurodegeneration by augmenting HDAC6 to enhance autophagy.     

A ubiquitin-like system mediates protein lipidation

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.     

美洲大蠊多肽和顺铂联用对HepG2细胞凋亡及自噬的影响

采用MTT法检测美洲大蠊多肽（PAP-3）与不同浓度的顺铂（DDP）单用或联合使用对人肝癌HepG2细胞增殖的影响；采用流式细胞术检测PAP-3、DDP单独或联合给药干预对HepG2细胞凋亡的影响；Western Blot检测PAP-3、DDP单独或联合给药后HepG2细胞内自噬相关蛋白p62、LC3、Beclin-1、Atg5及PI3K的表达。实验结果显示，与DDP单独给药相比，PAP-3与DDP联合给药对HepG2的细胞的抑制作用更强；PAP-3与DDP联合给药组中细胞的凋亡率与DDP和PAP-3单独给药组相比均有升高（P ＜ 0.05)。与对照组相比，DDP组中，p62蛋白水平降低，LC3II蛋白水平升高，LC3II/I的比例也升高，表现出细胞自噬流的活化；与DDP组相比，联合给药组中p62蛋白水平回升，LC3II蛋白水平和LC3II/I的比例均有回落。且联合给药组中PI3K、Atg5、Beclin-1等自噬相关蛋白的量均较DDP组减少，而凋亡相关的Caspase-3蛋白的表达量则较DDP组增加。据此推测，PAP-3和DDP的联用可以通过抑制HepG2细胞自噬相关蛋白的表达水平，抑制DDP带来的细胞自噬水平的升高，增加细胞对DDP的敏感性，从而诱导HepG2细胞凋亡。     

Ambra1 regulates autophagy and development of the nervous system

Autophagy is a self-degradative process involved both in basal turnover of cellular components and in response to nutrient starvation or organelle damage in a wide range of eukaryotes. During autophagy, portions of the cytoplasm are sequestered by double-membraned vesicles called autophagosomes, and are degraded after fusion with lysosomes for subsequent recycling. In vertebrates, this process acts as a pro-survival or pro-death mechanism in different physiological and pathological conditions, such as neurodegeneration and cancer; however, the roles of autophagy during embryonic development are still largely uncharacterized. Beclin1 (Becn1; coiled-coil, myosin-like BCL2-interacting protein) is a principal regulator in autophagosome formation, and its deficiency results in early embryonic lethality. Here we show that Ambra1 (activating molecule in Beclin1-regulated autophagy), a large, previously unknown protein bearing a WD40 domain at its amino terminus, regulates autophagy and has a crucial role in embryogenesis. We found that Ambra1 is a positive regulator of the Becn1-dependent programme of autophagy, as revealed by its overexpression and by RNA interference experiments in vitro. Notably, Ambra1 functional deficiency in mouse embryos leads to severe neural tube defects associated with autophagy impairment, accumulation of ubiquitinated proteins, unbalanced cell proliferation and excessive apoptotic cell death. In addition to identifying a new and essential element regulating the autophagy programme, our results provide in vivo evidence supporting the existence of a complex interplay between autophagy, cell growth and cell death required for neural development in mammals.     

NVP-BEZ235诱导自噬在肝癌细胞凋亡中的作用研究

目的 以Hep3B和PLC/PRF/5两种肝癌细胞为研究对象,探讨自噬在NVP-BEZ235中引起细胞凋亡的作用.方法 人类肝癌细胞Hep3B和PLC/PRF/5在含有10%FBS的DMEM培养基中进行培养,然后再进行细胞活性检测,用于Western blotting检测及线粒体膜电位(MMP)分析.结果 NVP-BEZ235能够有效增加LC3-Ⅱ的表达,并同时降低p62的表达,由此推断,NVP-BEZ235也能够诱导产生自噬,与Atg5的siRNA或自噬抑制剂3-甲基腺嘌呤(3-MA)联合应用时,肝癌细胞的生长被抑制.结论 NVP-BEZ235与Atg5的siRNA或者3-MA的联合应用能够促进细胞凋亡,NVP-BEZ235和自噬抑制剂的联合应用可为肝癌和其他恶性肿瘤临床治疗提供新的方法.     

Induction of autophagy and inhibition of tumorigenesis by beclin 1

The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.     

Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death

Huntington's disease is caused by an abnormal polyglutamine expansion within the protein huntingtin and is characterized by microscopic inclusion bodies of aggregated huntingtin and by the death of selected types of neuron. Whether inclusion bodies are pathogenic, incidental or a beneficial coping response is controversial. To resolve this issue we have developed an automated microscope that returns to precisely the same neuron after arbitrary intervals, even after cells have been removed from the microscope stage. Here we show, by survival analysis, that neurons die in a time-independent fashion but one that is dependent on mutant huntingtin dose and polyglutamine expansion; many neurons die without forming an inclusion body. Rather, the amount of diffuse intracellular huntingtin predicts whether and when inclusion body formation or death will occur. Surprisingly, inclusion body formation predicts improved survival and leads to decreased levels of mutant huntingtin elsewhere in a neuron. Thus, inclusion body formation can function as a coping response to toxic mutant huntingtin.     

Molecular cloning and expression of brain-derived neurotrophic factor

During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.     

Motor neuron columnar fate imposed by sequential phases of Hox-c activity

The organization of neurons into columns is a prominent feature of central nervous system structure and function. In many regions of the central nervous system the grouping of neurons into columns links cell-body position to axonal trajectory, thus contributing to the establishment of topographic neural maps. This link is prominent in the developing spinal cord, where columnar sets of motor neurons innervate distinct targets in the periphery. We show here that sequential phases of Hox-c protein expression and activity control the columnar differentiation of spinal motor neurons. Hox expression in neural progenitors is established by graded fibroblast growth factor signalling and translated into a distinct motor neuron Hox pattern. Motor neuron columnar fate then emerges through cell autonomous repressor and activator functions of Hox proteins. Hox proteins also direct the expression of genes that establish motor topographic projections, thus implicating Hox proteins as critical determinants of spinal motor neuron identity and organization.     

GST融合蛋白包涵体纯化方法的探索

克隆结核分枝杆菌培养滤液蛋白CFP10基因,并在大肠杆菌中进行表达和纯化。用PCR方法从结核分枝杆菌H37Rv基因组扩增出CFP10基因片段,克隆至pMD18-T载体中,序列测定正确后,将其亚克隆到表达载体pGEX-4T-1并在大肠杆菌BL21中表达,表达蛋白经SDS-PAG及Western-blot分析后,亲和层析法纯化蛋白。成功克隆了CFP10基因,并对其在E.coli中进行了表达,SDS-PAGE及Western blot分析表明表达产物正确。通过GST纯化系统获得36kD纯化蛋白,与文献报道相符,该蛋白可与CFP10 mAb特异结合,并且同时与活动期结核病人血清发生反应。成功获得了纯化的CFP10蛋白,为进一步研究CFP10蛋白的致病机理提供了实验依据。     

包涵体复性研究

包涵体的形成是外源重组蛋白质在大肠杆菌中高效表达的必然结果，也是目前产生重组蛋白质最有效的方法之一、综述了国内外在外源重组蛋白复性研究方面的主要进展，包括包涵体的分离和溶解、外源重组蛋白复性的原则、影响复性的物理因素及各种复性方法．     

Localization of nitric oxide synthase indicating a neural role for nitric oxide.

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum. Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis. It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone. We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme. We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations. NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells. These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons.     

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.     

Transient aggregation of ubiquitinated proteins during dendritic cell maturation

Dendritic cells (DCs) are antigen-presenting cells with the unique capacity to initiate primary immune responses. Dendritic cells have a remarkable pattern of differentiation (maturation) that exhibits highly specific mechanisms to control antigen presentation restricted by major histocompatibility complex (MHC). MHC class I molecules present to CD8(+) cytotoxic T cells peptides that are derived mostly from cytosolic proteins, which are ubiquitinated and then degraded by the proteasome. Here we show that on inflammatory stimulation, DCs accumulate newly synthesized ubiquitinated proteins in large cytosolic structures. These structures are similar to, but distinct from, aggresomes and inclusion bodies observed in many amyloid diseases. Notably, these dendritic cell aggresome-like induced structures (DALIS) are transient, require continuous protein synthesis and do not affect the ubiquitin-proteasome pathway. Our observations suggest the existence of an organized prioritization of protein degradation in stimulated DCs, which is probably important for regulating MHC class I presentation during maturation.