Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy -autophagosome formation and maturation -but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.© 2008 Macmillan Publishers Limited. All rights reserved. 6 Correspondence should be addressed to J.U.J. (jaeujung@usc.edu). AUTHOR CONTRIBUTIONS C.L. performed all aspects of this study; L.S., K.I., M.G., Q.L. and P.F. assisted with the experimental design and in collecting the data; E.R., I.V. and V.D. assisted with the autophagic protein degradation and in vitro endosome fusion assay; C.A. provided Vps constructs and their antibodies; C.L. and J.J. organized this study and wrote the paper. All authors discussed the results and commented on the manuscript.Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 May 31. Published in final edited form as:Nat Cell Biol. 2008 July ; 10(7): 776-787. doi:10.1038/ncb1740. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAutophagy is a tightly regulated membrane rearrangement process that ensures lysosomedependent bulk degradation of cytosolic proteins or organelles, and is highly conserved in eukaryotic cells, as seen with the endocytic pathway 1 . In response to environmental stresses, portions of cytoplasmic constituents are engulfed by a unique membrane structure, the phagophore, as it elongates to form a double-or multiple-membrane-bound compartment called the autophagosome. Newly synthesized autophagosomes then undergo extensive remodelling to acquire degradative capabilities. The remodelling process, also known as autophagosomal maturation, involves sequential fusion of autophagosomes with endocytic vesicles...

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Five N-methyl-D-aspartate (NMDA) receptor subunits have been identified thus far: NR1, NR2A, NR2B, NR2C, and NR2D. Here, we have analyzed the expression patterns of mRNAs for the NMDA receptor subunits in the developing and adult rats by in situ hybridization. The developmental changes of the expression patterns were most salient in the cerebellum. In the external granular layer, hybridization signals of mRNAs for NR1, NR2A, NR2B, and NR2C appeared by postnatal day 3, but no NR2D mRNA was expressed at any developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stage examined. The NR1 mRNA was expressed in all cerebellar neurons at all developmental stages examined. The signals for the NR2A mRNA appeared in Purkinje cells and granule cells during the second postnatal week. The signals for the NR2B mRNA in granule cells were seen transiently during the first 2 weeks after birth. The signals for NR2C mRNA appeared in granule cells and glial cells during the second postnatal week. The signals for NR2D mRNA appeared transiently in Purkinje cells during the first 8 postnatal days; in adult rats, these were seen in stellate and Golgi cells. In the cerebellar nuclei, mRNAs for NR1, NR2A, NR2B, and NR2D were more or less expressed on postnatal day 0, while expression signals for the NR2C mRNA were first detected in postnatal day 14. Thus, the most conspicuous changes of expression patterns were observed in the cerebellar cortex during the first 2 weeks after birth, when development and maturation of the cerebellum proceed most rapidly.

We report the molecular characterization of a mouse basic helix-loop-helix factor, designated MATH-1, structurally related to the product of the Drosophila proneural gene atonal. MATH-1 mRNA is first detected in the cranial ganglions and the dorsal part of the central nervous system on embryonic day 9.5 (E9.5). From E10.5 onward, prominent expression of MATH-1 continues in the dorsal part of the central nervous system but becomes restricted to the external granular layer of the cerebellum by E18 and is undetectable in the adult nervous system. MATH-1 activates E box-dependent transcription in collaboration with E47, but the activity is completely antagonized by the negative regulator of neurogenesis HES-1. These results suggest that MATH-1 may be a target of HES-1 and play a role in the differentiation of subsets of neural cells by activating E box-dependent transcription.

We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o-protein coupled P2Y(12) receptor (P2Y(12)R). Here we report discovering that the P2X(4) receptor (P2X(4)R) is also involved in ATP-induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y(12)R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3'-kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP-induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y(12)R antagonist. These results indicate that P2Y(12)R-mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X(4)R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X(4)R significantly inhibited it. Knockdown of the P2X(4) receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X(4)R as well as P2Y(12)R is involved in ATP-induced microglial chemotaxis.