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Identification of a molecular target of psychosine and its role in globoid cell formation.

Im DS, Heise CE, Nguyen T, O'Dowd BF, Lynch KR - J. Cell Biol. (2001)

Bottom Line: The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase.Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor.Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.

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Inhibition of cytokinesis by PSY. TDAG8-RH7777 (A) or TDAG8-HEK293 (B) cells were stained with DAPI (middle). Green lines on overlays indicate boundaries of single cells. (C) Representative FACS® data from HEK293 cells (top) or HEK293 cells expressing TDAG8 (bottom) treated either with 10 μM PSY (C-2 and C-4) or vehicle (C-1 and C-3). Cell cultures were treated for 6 d.
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Figure 5: Inhibition of cytokinesis by PSY. TDAG8-RH7777 (A) or TDAG8-HEK293 (B) cells were stained with DAPI (middle). Green lines on overlays indicate boundaries of single cells. (C) Representative FACS® data from HEK293 cells (top) or HEK293 cells expressing TDAG8 (bottom) treated either with 10 μM PSY (C-2 and C-4) or vehicle (C-1 and C-3). Cell cultures were treated for 6 d.

Mentions: To test the hypothesis that PSY acting via TDAG8 mediates the disjunction of mitogenesis and cytokinesis characteristic of globoid cells, we treated cell cultures transfected with TDAG8 DNA with PSY and quantified nuclear DNA content. When TDAG8-expressing RH7777 cells were treated with 10 μM PSY, multinuclear globoid cells were observed by DAPI staining (Fig. 5 A, and Table ). Neither expression of TDAG8 without PSY treatment nor PSY treatment in the absence of TDAG8 DNA transfection resulted in the appearance of multinuclear, globoid cells. Likewise, TDAG8/HEK293 cells became multinuclear in response to PSY treatment (Fig. 5B and Fig. c) and both receptor and ligand were required to generate the globoid cell phenotype. In concert with the structure activity profile found with inhibition of cAMP and calcium mobilization (see above), GlcPSY and lysosulfatide mimicked the action of PSY in evoking globoid cell formation, whereas N-acetyl PSY and SPC did not (data not shown). The requirement for both members of the ligand receptor pair to be present, the long time course required for globoid cell formation, and a previous report using U937 cells (Kanazawa et al. 2000) all suggest that this phenomenon involves a disjunction of mitosis and cytokinesis rather than simple cell fusion.


Identification of a molecular target of psychosine and its role in globoid cell formation.

Im DS, Heise CE, Nguyen T, O'Dowd BF, Lynch KR - J. Cell Biol. (2001)

Inhibition of cytokinesis by PSY. TDAG8-RH7777 (A) or TDAG8-HEK293 (B) cells were stained with DAPI (middle). Green lines on overlays indicate boundaries of single cells. (C) Representative FACS® data from HEK293 cells (top) or HEK293 cells expressing TDAG8 (bottom) treated either with 10 μM PSY (C-2 and C-4) or vehicle (C-1 and C-3). Cell cultures were treated for 6 d.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169470&req=5

Figure 5: Inhibition of cytokinesis by PSY. TDAG8-RH7777 (A) or TDAG8-HEK293 (B) cells were stained with DAPI (middle). Green lines on overlays indicate boundaries of single cells. (C) Representative FACS® data from HEK293 cells (top) or HEK293 cells expressing TDAG8 (bottom) treated either with 10 μM PSY (C-2 and C-4) or vehicle (C-1 and C-3). Cell cultures were treated for 6 d.
Mentions: To test the hypothesis that PSY acting via TDAG8 mediates the disjunction of mitogenesis and cytokinesis characteristic of globoid cells, we treated cell cultures transfected with TDAG8 DNA with PSY and quantified nuclear DNA content. When TDAG8-expressing RH7777 cells were treated with 10 μM PSY, multinuclear globoid cells were observed by DAPI staining (Fig. 5 A, and Table ). Neither expression of TDAG8 without PSY treatment nor PSY treatment in the absence of TDAG8 DNA transfection resulted in the appearance of multinuclear, globoid cells. Likewise, TDAG8/HEK293 cells became multinuclear in response to PSY treatment (Fig. 5B and Fig. c) and both receptor and ligand were required to generate the globoid cell phenotype. In concert with the structure activity profile found with inhibition of cAMP and calcium mobilization (see above), GlcPSY and lysosulfatide mimicked the action of PSY in evoking globoid cell formation, whereas N-acetyl PSY and SPC did not (data not shown). The requirement for both members of the ligand receptor pair to be present, the long time course required for globoid cell formation, and a previous report using U937 cells (Kanazawa et al. 2000) all suggest that this phenomenon involves a disjunction of mitosis and cytokinesis rather than simple cell fusion.

Bottom Line: The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase.Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor.Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.

Show MeSH
Related in: MedlinePlus