Biomedical subjects

Bor Luen Tang

Publications and source records attributed to Bor Luen Tang.

34 records · Page 2Linked to original sources

PubMed2004

Participation of the syntaxin 5/Ykt6/GS28/GS15 SNARE complex in transport from the early/recycling endosome to the trans-Golgi network.

An in vitro transport assay, established with a modified Shiga toxin B subunit (STxB) as a marker, has proved to be useful for the study of transport from the early/recycling endosome (EE/RE) to the trans-Golgi network (TGN). Here, we modified this assay to test antibodies to all known soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that have been shown to localize in the Golgi and found that syntaxin 5, GS28, Ykt6, and GS15 antibodies specifically inhibited STxB transport. Because syntaxin 5, GS28, Ykt6, and GS15 exist as a unique SNARE complex, our observation indicates that these four SNAREs function as a complex in EE/RE-TGN transport. The importance of GS15 in EE/RE-TGN transport was further demonstrated by a block in recombinant STxB transport in HeLa cells when GS15 expression was knocked down by its small interfering iRNA. Morphological analyses showed that some GS15 and Ykt6 were redistributed from the Golgi to the endosomes when the recycling endosome was perturbed by SNX3-overexpression, suggesting that GS15 and Ykt6 might cycle between the endosomes and the Golgi apparatus. Further studies indicated that syntaxin 5 and syntaxin 16 exerted their role in EE/RE-TGN transport in an additive manner. The kinetics of inhibition exhibited by syntaxin 16 and syntaxin 5 antibodies is similar.

Animals↗

PubMed2004

Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions.

Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.

Animals↗

PubMed2004

Structural characterization of the human Nogo-A functional domains. Solution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration.

The recent discovery of the Nogo family of myelin inhibitors and the Nogo-66 receptor opens up a very promising avenue for the development of therapeutic agents for treating spinal cord injury. Nogo-A, the largest member of the Nogo family, is a multidomain protein containing at least two regions responsible for inhibiting central nervous system (CNS) regeneration. So far, no structural information is available for Nogo-A or any of its structural domains. We have subcloned and expressed two Nogo-A fragments, namely the 182 residue Nogo-A(567-748) and the 66 residue Nogo-66 in Escherichia coli. CD and NMR characterization indicated that Nogo-A(567-748) was only partially structured while Nogo-66 was highly insoluble. Nogo-40, a truncated form of Nogo-66, has been previously shown to be a Nogo-66 receptor antagonist that is able to enhance CNS neuronal regeneration. Detailed NMR examinations revealed that a Nogo-40 peptide had intrinsic helix-forming propensity, even in an aqueous environment. The NMR structure of Nogo-40 was therefore determined in the presence of the helix-stabilizing solvent trifluoroethanol. The solution structure of Nogo-40 revealed two well-defined helices linked by an unstructured loop, representing the first structure of Nogo-66 receptor binding ligands. Our results provide the first structural insights into Nogo-A functional domains and may have implications in further designs of peptide mimetics that would enhance CNS neuronal regeneration.

Amino Acid Sequence↗

PubMed2004

Inter- and intracellular interactions of Nogo: new findings and hypothesis.

The molecule Nogo has captured the imagination of many as a possible key player, and therefore therapeutic target, in the pathological settings of central nervous system (CNS) injury and degenerative pathology. Found in both glial cells and neurons, the endogenous, physiological role of Nogo is as yet unknown. Recently reported targeted disruption of the Nogo gene did not result in any obvious neuro-anatomical or neurological phenotype. Compared with wild-type mice, Nogo-deficient mice also did not exhibit a truly convincing enhancement in their ability to regenerate CNS neurons upon injury. Does the molecule have any important physiological function at all? Other recent discoveries of new interacting partners of Nogo at the mitochondria and the CNS paranode suggest intriguing links to the modulation of apoptosis and developmental organization or signalling at the axoglial junction.

Animals↗

PubMed2004

Nogo-66 and myelin-associated glycoprotein (MAG) inhibit the adhesion and migration of Nogo-66 receptor expressing human glioma cells.

Malignant gliomas are common and aggressive brain tumours associated with significant morbidity and mortality. We showed in this report that substratum adherence and migration by human U87MG glioma cells in culture were significantly attenuated by the extracellular domains of Nogo-A (Nogo-66) and the myelin-associated glycoprotein (MAG). U87MG cells contained significant amounts of endogenous Nogo-66 receptor (NgR), and treatment of the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) or NgR antibodies resulted in an increase in their ability to adhere to, or migrate through, Nogo-66- and MAG-coated substrates. Nogo-66 and MAG may therefore modulate glioma growth and migration by acting through the NgR, a phenomenon that has potential therapeutic implications.

Antibodies↗

PubMed2003

Nogo-A at CNS paranodes is a ligand of Caspr: possible regulation of K(+) channel localization.

We report Nogo-A as an oligodendroglial component congregating and interacting with the Caspr-F3 complex at paranodes. However, its receptor Nogo-66 receptor (NgR) does not segregate to specific axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound specifically to substrates coated with Nogo-66 peptide and GST-Nogo-66. Binding persisted even after phosphatidylinositol- specific phospholipase C (PI-PLC) removal of GPI-linked F3 from the cell surface, suggesting a direct interaction between Nogo-66 and Caspr. Both Nogo-A and Caspr co-immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo-A-Caspr and K(+) channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT(-/-) mice), distances between the paired labeling of K(+) channels were shortened significantly and their localization shifted toward paranodes, while paranodal Nogo-A congregation was markedly reduced. Our results demonstrate that Nogo-A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon-glial junction architecture and possibly K(+)-channel localization during development.

Amino Acid Sequence↗

PubMed2003

Inhibitors of neuronal regeneration: mediators and signaling mechanisms.

Neuritogenesis and its inhibition are opposite and balancing processes during development as well as pathological states of adult neuron. In particular, the inability of adult central nervous system (CNS) neurons to regenerate upon injury has been attributed to both a lack of neuritogenic ability and the presence of neuronal growth inhibitors in the CNS environment. I review here recent progress in our understanding of neuritogenic inhibitors, with particular emphasis on those with a role in the inhibition of neuronal regeneration in the CNS, their signaling cascades and signal mediators. Neurotrophines acting through the tropomyosin-related kinase (Trk) family and p75 receptors promote neuritogenesis, which appears to require sustained activation of the mitogen activated protein (MAP) kinase pathway, and/or the activation of phosphotidylinositol 3-kinase (PI3 kinase). During development, a plethora of guidance factors and their receptors navigate the growing axon. However, much remained to be learned about the signaling receptors and pathways that mediate the activity of inhibitors of CNS regeneration. There is growing evidence that neuronal guidance molecules, particularly semaphorins, may also have a role as inhibitors of CNS regeneration. Although direct links have not yet been established in many cases, signals from these agents may ultimately converge upon the modulators and effectors of the Rho-family GTPases. Rho-family GTPases and their effectors modulate the activities of actin modifying molecules such as cofilin and profilin, resulting in cytoskeletal changes associated with growth cone extension or retraction.

Adult↗

PubMed2002

Ultrastructural characterization of endoplasmic reticulum--Golgi transport containers (EGTC).

Recent observations made in live cells expressing green fluorescent protein (GFP)-tagged cargo markers have demonstrated the existence of large, mobile transport intermediates linking peripheral ER exit sites (ERES) to the perinuclear Golgi. Using a procedure of rapid ethane freezing, we examined ultrastructurally the intermediates involved in ER-Golgi transport of the vesicular stomatitis virus (VSV) G protein. When released at the permissive temperature of 32 degrees C, VSVG is first found to be concentrated in pleiomorphic, membrane-bound structures (of about 0.4 to 1 microm in diameter) with extensive budding profiles. These structures are devoid of COPII components and Golgi markers, but are enriched in COPI, the retrograde cargo ERGIC53, and the tethering protein p115. The structures appear to be able to undergo fusion with the Golgi stack and are tentatively referred to as ER-Golgi transport containers, or EGTCs. VSVG protein exiting the ERES at 15 degrees C is first found in clusters or strings of COPII-containing small vesicles, and morphological analysis indicates that these clusters and strings of COPII vesicles may coalesce by homotypic fusion to form the EGTCs. Together with the large transport containers mediating transport from the trans-Golgi network to the plasma membrane, EGTCs represents an emerging class of large membranous structures mediating anterograde transport between the major stations of the exocytic pathway.

Animals↗

PubMed2002

Nogo-A expression in mouse central nervous system neurons.

The longest central nervous system (CNS) specific isoform of the major myelin-associated inhibitor of neurite growth, Nogo-A, has previously been shown to be expressed largely in oligodendrocytes. Using an antibody raised against a recombinant fusion protein comprising amino acids 223-399 of Nogo-A, we show in this report that Nogo-A is also expressed in the cell body of a distinct set of CNS neurons. The antibody detects a single protein band of 220 kDa in rat brain lysate and neuroblastoma cell lysates. Immunofluorescent analyses reveal that Nogo-A is found largely in the endoplasmic reticulum of neuroblastoma cell lines SH-SY5Y and NIE-115. In the mouse CNS, Nogo-A can be found in specific subsets of neuronal cell bodies in addition to oligodendrocytes, but not glial fibrillary associated protein positive astrocytes or MAC-1 positive microglia. Our results provide a conclusive demonstration of the expression of Nogo-A in CNS neurons, which suggests that Nogo-A may have distinct endogenous roles in neurons other than its known ability to inhibit neurite growth.

Animals↗

PubMed2002

Interferon-beta administration confers a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia.

We demonstrate that treatment with the cytokine human interferon-beta (IFN-beta) resulted in a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia. Rabbits administered subcutaneously with IFN-beta prior to (pre-treatment) or after (post-treatment) the introduction of an autologous blood clot into the middle cerebral artery have consistently smaller subcortical infarct volumes compared to control (untreated) counterparts. The average subcortical infarct volume of pre-treatment rabbits is 46.3+/-9.3 mm(3) (n=4), and that of post-treatment rabbit is 40.0+/-23.1 mm(3) (n=4). Both are significantly lower (P=0.003 and P=0.004, respectively) than the average subcortical infarct volume of control rabbits (121.6+/-32.9 mm(3), n=5). Although the precise mechanism responsible for the beneficial effect of IFN-beta on stroke is not yet clear, our results are in line with the known anti-inflammatory potential and anti-apoptotic function of IFN-beta.

Animals↗

PubMed2002

Neuritogenesis: polarization of constitutive exocytosis by effectors of Rho-family GTPases?

The sprouting of neurites from a neuron represents a highly specialized form of cellular morphogenesis that must involve coordinated changes in two major cellular processes at two membrane locations: reorganization of the cytoskeleton and redirection of membrane traffic from the trans-Golgi network to the plasma membrane of the growth tip. How exactly are these two processes linked and how is spatial and temporal coordination achieved at the first instance of neurite sprouting? Recent advances may have already revealed some, if not most of the pieces in the puzzle. We discuss below, with some extrapolations, of what has recently come to light, and what more is needed to construct a coherent picture.

Animals↗

PubMed2002

Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration.

The recently cloned gene Nogo, whose alternative splice products correspond to the antigenic target of the central nervous system (CNS) regeneration enhancing monoclonal antibody IN-1, codes for membrane proteins enriched in brain, particularly in oligodendrocytes. The 66-amino acid extracellular domain of Nogo (Nogo-66) interacts with a high-affinity receptor (NgR), a glycosylphosphatidylinositol (GPI)-linked protein with multiple leucine-rich repeats. The amino terminal cytoplasmic domain of Nogo appears to have a general cellular growth inhibitory effect. Nogo-66, on the other hand, specifically retards neurite outgrowth and induces growth cone collapse, possibly through its interaction with NgR and as yet unidentified transmembrane coreceptors. Recent results also suggest that Nogo expression may induce apoptosis in tumor cells. Together, these proteins provide new molecular handles for the design of therapeutic interventions for CNS injuries and neurodegenerative diseases, as well as possible leads to anticancer strategies.

Animals↗

PubMed2002

Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform.

The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.

Animals↗

PubMedDate not supplied

Trans-Golgi network syntaxin 10 functions distinctly from syntaxins 6 and 16.

Syntaxin 10 is a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein localized to the trans-Golgi network (TGN), where two other members of the syntaxin family, syntaxins 6 and 16, also reside. The role of syntaxin 10 in regulating TGN protein traffic is not yet defined. Syntaxin 10 co-localizes well with syntaxins 6 and 16 at the TGN in interphase cells, and appears to interact with both syntaxin 6 and 16 as evidenced by co-immunoprecipitation analyses. However, unlike syntaxin 6 and 16, neither syntaxin 10 antibodies nor its cytosolic domain inhibits endosome-TGN transport of shiga toxin. Syntaxin 16 knockdown with small interfering RNA (siRNA) affects the TGN localization of syntaxin 6 but not syntaxin 10, and clearly inhibits endosome-TGN transport. On the other hand, knockdown of syntaxin 10 expressions had no significant effect on the TGN localization of syntaxin 6 and 16, and did not inhibit endosome-TGN transport. Unlike syntaxin 16, syntaxin 10 does not interact specifically with Vps45, the Sec1/Munc18 (SM) family member at the TGN. On the other hand, syntaxin 10 reciprocally co-immunoprecipitated endosomal syntaxin 12/13, and knockdown of syntaxin 10 expressions affects the surface expression of transferrin receptor (TfR) and seems to induce the formation of an immobile TfR pool. Therefore, in spite of its co-localization and possible interaction with syntaxin 16, syntaxin 10 is not part of the syntaxin 16-based SNARE complex involved in endosome-TGN transport, and may have a hitherto unrecognized function in the TGN-endosome boundary.

Animals↗

PubMedDate not supplied

SNAREs in neurons--beyond synaptic vesicle exocytosis (Review).

The paradigm for soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) function in mammalian cells has been built on advancements in our understanding of structural and biochemical aspects of synaptic vesicle exocytosis, involving specifically synaptobrevin, syntaxin 1 and SNAP25. Interestingly, a good number of SNAREs which are not directly involved in neurotransmitter exocytosis, are either brain-enriched or have distinct neuron-specific functions. Syntaxins 12/13 regulates glutamate receptor recycling via its interaction with neuron-enriched endosomal protein of 21 kDa (NEEP21). TI-VAMP/VAMP7 is essential for neuronal morphogenesis and mediates the vesicular transport processes underlying neurite outgrowth. Ykt6 is highly enriched in the cerebral cortex and hippocampus and is targeted to a novel compartment in neurons. Syntaxin 16 has a moderate expression level in many tissues, but is rather enriched in the brain. Here, we review and discuss the neuron-specific physiology and possible pathology of these and other (such as SNAP-29 and Vti1a-beta) members of the SNARE family.

Animals↗

PubMedDate not supplied

alpha-synuclein and Parkinson's disease: the first roadblock.

alpha-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and alpha-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of alpha-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed alpha- synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. alpha-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that alpha- synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of alpha-synuclein-induced neurodegeneration. alpha-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of alpha-synuclein with components of neuronal membrane traffic.

Animals↗