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1.
Autophagy ; 14(5): 764-777, 2018.
Article in English | MEDLINE | ID: mdl-28513333

ABSTRACT

Conditional knockout mice for Atg9a, specifically in brain tissue, were generated to understand the roles of ATG9A in the neural tissue cells. The mice were born normally, but half of them died within one wk, and none lived beyond 4 wk of age. SQSTM1/p62 and NBR1, receptor proteins for selective autophagy, together with ubiquitin, accumulated in Atg9a-deficient neurosoma at postnatal d 15 (P15), indicating an inhibition of autophagy, whereas these proteins were significantly decreased at P28, as evidenced by immunohistochemistry, electron microscopy and western blot. Conversely, degenerative changes such as spongiosis of nerve fiber tracts proceeded in axons and their terminals that were occupied with aberrant membrane structures and amorphous materials at P28, although no clear-cut degenerative change was detected in neuronal cell bodies. Different from autophagy, diffusion tensor magnetic resonance imaging and histological observations revealed Atg9a-deficiency-induced dysgenesis of the corpus callosum and anterior commissure. As for the neurite extensions of primary cultured neurons, the neurite outgrowth after 3 d culturing was significantly impaired in primary neurons from atg9a-KO mouse brains, but not in those from atg7-KO and atg16l1-KO brains. Moreover, this tendency was also confirmed in Atg9a-knockdown neurons under an atg7-KO background, indicating the role of ATG9A in the regulation of neurite outgrowth that is independent of autophagy. These results suggest that Atg9a deficiency causes progressive degeneration in the axons and their terminals, but not in neuronal cell bodies, where the degradations of SQSTM1/p62 and NBR1 were insufficiently suppressed. Moreover, the deletion of Atg9a impaired nerve fiber tract formation.


Subject(s)
Autophagy-Related Proteins/deficiency , Axons/metabolism , Membrane Proteins/deficiency , Nerve Net/metabolism , Vesicular Transport Proteins/deficiency , Animals , Autophagy-Related Proteins/metabolism , Axons/ultrastructure , Cells, Cultured , Corpus Callosum/metabolism , Corpus Callosum/pathology , Integrases/metabolism , Intracellular Signaling Peptides and Proteins , Membrane Proteins/metabolism , Mice, Knockout , Neurites/metabolism , Neurites/ultrastructure , Phenotype , Proteins/metabolism , Purkinje Cells/metabolism , Purkinje Cells/ultrastructure , Sequestosome-1 Protein/metabolism , Vesicular Transport Proteins/metabolism
2.
J Cell Sci ; 129(2): 329-40, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26627821

ABSTRACT

Rab GTPases act as molecular switches regulating various aspects of membrane trafficking. Among them, Rab5 and Rab7 play central roles in the endolysosomal network. Although many effectors downstream of Rab7 have been elucidated, our present understanding of the mechanism regulating Rab7 activity is extremely limited. It has only recently been accepted that the Mon1-Ccz1 complex is a Rab7 guanine nucleotide exchange factor, but it still remains unclear what the location where Mon1-Ccz1 works with Rab7 is. To address what kind of change or switch exists in the regulatory mechanism upstream of Rab7 during its transition from the late endosome to lysosome, we examined Rab7 activity in steady-state cells and during EGF-induced macropinocytosis using a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1-Ccz1 and is implicated in late-endosome-lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1-Ccz1 and active Rab7 played a role in perinuclear clustering of lysosomes.


Subject(s)
Endosomes/enzymology , Lysosomes/enzymology , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , Enzyme Activation , HEK293 Cells , HeLa Cells , Humans , Metabolic Networks and Pathways , Protein Transport , rab7 GTP-Binding Proteins
3.
Biomed Res ; 36(2): 121-33, 2015.
Article in English | MEDLINE | ID: mdl-25876663

ABSTRACT

Autophagy is essential for the maintenance of cellular metabolism. Once autophagy is induced in cells, the isolation membrane forms a so-called phagophore. The endoplasmic reticulum (ER) is one of several candidates for the membrane source for phagophores. Recently, LC3-positive isolation membranes were found to emerge from a DFCP1 (double FYVE domain-containing protein)-positive, ER-associated compartment called the omegasome. Although the GFP-tagged DFCP1 protein has been examined in cultured cells, little is known about the precise cellular and tissue distribution of this endogenous protein. To determine the expression of the endogenous DFCP1 protein, we produced antibodies specific to mouse DFCP1 protein. The antibody recognized both human and mouse DFCP1 proteins, both of which have molecular masses of approximately 87 kDa. In HeLa cells under normal conditions, immunoreactivity for DFCP1 was found dotted or tubular along Tom20-positive filamentous mitochondria and was only partially co-localized in the ER or Golgi apparatus. Moreover, under starved conditions, distinct DFCP1-positive structures became more dotted and scattered in the cytoplasm, while one part of the LC3-positive autophagosomes were immunopositive for DFCP1. These results indicate that an antibody raised against DFCP1 could be a useful tool in explaining the mechanism of phagophore formation from omegasome compartments.


Subject(s)
Carrier Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies/chemistry , Carrier Proteins/chemistry , Carrier Proteins/immunology , Cerebral Cortex/cytology , Fluorescent Antibody Technique, Indirect , HeLa Cells , Humans , Male , Mice, Inbred C57BL , Molecular Sequence Data , Neurons/metabolism , Organ Specificity , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein Transport
4.
PLoS One ; 8(5): e63568, 2013.
Article in English | MEDLINE | ID: mdl-23671684

ABSTRACT

GABAA receptor-associated protein (GABARAP) was initially identified as a protein that interacts with GABAA receptor. Although LC3 (microtubule-associated protein 1 light chain 3), a GABARAP homolog, has been localized in the dendrites and cell bodies of neurons under normal conditions, the subcellular distribution of GABARAP in neurons remains unclear. Subcellular fractionation indicated that endogenous GABARAP was localized to the microsome-enriched and synaptic vesicle-enriched fractions of mouse brain as GABARAP-I, an unlipidated form. To investigate the distribution of GABARAP in neurons, we generated GFP-GABARAP transgenic mice. Immunohistochemistry in these transgenic mice showed that positive signals for GFP-GABARAP were widely distributed in neurons in various brain regions, including the hippocampus and cerebellum. Interestingly, intense diffuse and/or fibrillary expression of GFP-GABARAP was detected along the axonal initial segments (AIS) of hippocampal pyramidal neurons and cerebellar Purkinje cells, in addition to the cell bodies and dendrites of these neurons. In contrast, only slight amounts of LC3 were detected along the AIS of these neurons, while diffuse and/or fibrillary staining for LC3 was mainly detected in their cell bodies and dendrites. These results indicated that, compared with LC3, GABARAP is enriched in the AIS, in addition to the cell bodies and dendrites, of these hippocampal pyramidal neurons and cerebellar Purkinje cells.


Subject(s)
Axons/metabolism , Cytoskeletal Proteins/metabolism , Membrane Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Neurons/metabolism , Animals , Apoptosis Regulatory Proteins , Cerebellum/cytology , Cerebellum/metabolism , Cytoskeletal Proteins/genetics , Dendrites/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Immunoblotting , Immunohistochemistry , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Microscopy, Confocal , Microsomes/metabolism , Purkinje Cells/metabolism , Pyramidal Cells/metabolism , Synaptic Vesicles/metabolism
5.
Glycoconj J ; 29(1): 47-55, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22187327

ABSTRACT

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized type I membrane glycoprotein, and promotes dendritic filopodia formation. Although we have determined the N-glycan structures of ICAM-5 in a previous report, their function is unknown. Here, we produced fifteen ICAM-5 gene constructs, in which each potential N-glycosylation site was mutated, to elucidate the function of the N-glycans of ICAM-5, and observed the effects of transfection of them on a neuronal cell line, Neuro-2a (N2a). Only the N54Q mutant, which is the mutant for the most N-terminal glycosylation site, failed to induce filopodia-like protrusions in N2a cells. Immunofluorescence staining and cell surface biotinylation revealed that N54Q ICAM-5 was confined to the ER and also could not be expressed on the cell surface. This is further supported by the biochemical evidence that almost all N-glycans of N54Q ICAM-5 were digested by Endo glycosidase H and peptide:N-glycanase, indicating that almost all of them retain high-mannose-type structures in ER. In additon, it also failed to form disulfide bonds or functional protein complexes. The stable transformants of N54Q ICAM-5 showed retarded cell growth, but it was interesting that there was no apparent ER stress, because the mutant was sequentially degraded via ER associated degradation pathway by comparing the susceptibilities of the responses to various inhibitors of this pathway in wild-type and N54Q ICAM-5 transfectants. Taken together, the Asn(54)-linked glycan is necessary for normal trafficking and function of ICAM-5, but is unassociated with ER-associated degradation of it.


Subject(s)
Asparagine/metabolism , Cell Adhesion Molecules/metabolism , Nerve Tissue Proteins/metabolism , Polysaccharides/metabolism , Protein Folding , Animals , Asparagine/chemistry , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Endoplasmic Reticulum/metabolism , Glycoside Hydrolases/metabolism , Glycosylation , Humans , Immunohistochemistry , Mannose/metabolism , Mice , Microscopy, Fluorescence , Mutagenesis, Site-Directed/methods , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Polysaccharides/chemistry , Protein Processing, Post-Translational , Protein Transport , Pseudopodia/chemistry , Rats , Transfection/methods
6.
Cancer Res ; 70(1): 257-65, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-20048077

ABSTRACT

Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis.


Subject(s)
Apoptosis/drug effects , Radiation-Protective Agents/pharmacology , Tumor Suppressor Protein p53/drug effects , Vanadates/pharmacology , Animals , Benzothiazoles/pharmacology , Blotting, Western , Cell Line , Female , Flow Cytometry , Humans , Immunoprecipitation , Mice , Mice, Inbred ICR , Sulfonamides/pharmacology , Toluene/analogs & derivatives , Toluene/pharmacology , Transfection
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