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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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Related in: MedlinePlus

PSY induced Ca2+ mobilization in TDAG8-HEK293 cells. Cells were placed in suspension by treatment of monolayers with trypsin/EDTA, loaded with the calcium-sensitive fluorophore, INDO-1/AM, and 2–4 × 106 cells were placed in a temperature-controlled cuvette. (A) Representative traces in response to 10 μM PSY by TDAG8/HEK293 or mock-transfected HEK293 cells. (B) Concentration–response curve for PSY in evoking Ca2+ mobilization by TDAG8/HEK293 cells.
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Figure 2: PSY induced Ca2+ mobilization in TDAG8-HEK293 cells. Cells were placed in suspension by treatment of monolayers with trypsin/EDTA, loaded with the calcium-sensitive fluorophore, INDO-1/AM, and 2–4 × 106 cells were placed in a temperature-controlled cuvette. (A) Representative traces in response to 10 μM PSY by TDAG8/HEK293 or mock-transfected HEK293 cells. (B) Concentration–response curve for PSY in evoking Ca2+ mobilization by TDAG8/HEK293 cells.

Mentions: When expressed in RH7777 hepatoma cells, human TDAG8 mediated PSY-induced inhibition of forskolin-driven cAMP rise in a concentration-dependent manner (EC50 = 3.4 μM) (Fig. 1A and Fig. B). This response was evoked also by structurally related lysolipids, e.g., d-glucosyl-β-1,1′ sphingosine (GlcPSY), LacPSY, and lysosulfatide, but not by N-acetyl PSY, sphingosine 1-phosphate, lysophosphatidic acid, ceramide 1-phosphate, or lysophosphatidylcholine (Fig. 1 C). Similar results were found using the orthologous mouse TDAG8 DNA (data not shown). The PSY response was not blocked by pretreatment of RH7777 cultures with pertussis toxin (PTX; 100 ng/ml for 24 h), suggesting the involvement of PTX-insensitive G proteins, perhaps Gαz (Kozasa and Gilman 1995). SPC also was active in this assay, but this response, which was PTX sensitive (not shown), probably proceeds through an endogenous receptor in RH7777 cells (Im et al. 2000a). This suspicion was confirmed when SPC failed in further assays (see below). The ability of PSY to activate TDAG8 was confirmed by this lipid evoking Ca2+ transients in TDAG8/HEK293 cells (Fig. 2) and the PSY-driven internalization of a TDAG8/GFP fusion protein in HEK293T cells (Fig. 3). Both actions were mimicked by related lysoglycolipids (e.g., GlcPSY and lysosulfatide), but not by SPC or N-acetyl PSY at concentrations up to 10 μM (data not shown).


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)

PSY induced Ca2+ mobilization in TDAG8-HEK293 cells. Cells were placed in suspension by treatment of monolayers with trypsin/EDTA, loaded with the calcium-sensitive fluorophore, INDO-1/AM, and 2–4 × 106 cells were placed in a temperature-controlled cuvette. (A) Representative traces in response to 10 μM PSY by TDAG8/HEK293 or mock-transfected HEK293 cells. (B) Concentration–response curve for PSY in evoking Ca2+ mobilization by TDAG8/HEK293 cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169470&req=5

Figure 2: PSY induced Ca2+ mobilization in TDAG8-HEK293 cells. Cells were placed in suspension by treatment of monolayers with trypsin/EDTA, loaded with the calcium-sensitive fluorophore, INDO-1/AM, and 2–4 × 106 cells were placed in a temperature-controlled cuvette. (A) Representative traces in response to 10 μM PSY by TDAG8/HEK293 or mock-transfected HEK293 cells. (B) Concentration–response curve for PSY in evoking Ca2+ mobilization by TDAG8/HEK293 cells.
Mentions: When expressed in RH7777 hepatoma cells, human TDAG8 mediated PSY-induced inhibition of forskolin-driven cAMP rise in a concentration-dependent manner (EC50 = 3.4 μM) (Fig. 1A and Fig. B). This response was evoked also by structurally related lysolipids, e.g., d-glucosyl-β-1,1′ sphingosine (GlcPSY), LacPSY, and lysosulfatide, but not by N-acetyl PSY, sphingosine 1-phosphate, lysophosphatidic acid, ceramide 1-phosphate, or lysophosphatidylcholine (Fig. 1 C). Similar results were found using the orthologous mouse TDAG8 DNA (data not shown). The PSY response was not blocked by pretreatment of RH7777 cultures with pertussis toxin (PTX; 100 ng/ml for 24 h), suggesting the involvement of PTX-insensitive G proteins, perhaps Gαz (Kozasa and Gilman 1995). SPC also was active in this assay, but this response, which was PTX sensitive (not shown), probably proceeds through an endogenous receptor in RH7777 cells (Im et al. 2000a). This suspicion was confirmed when SPC failed in further assays (see below). The ability of PSY to activate TDAG8 was confirmed by this lipid evoking Ca2+ transients in TDAG8/HEK293 cells (Fig. 2) and the PSY-driven internalization of a TDAG8/GFP fusion protein in HEK293T cells (Fig. 3). Both actions were mimicked by related lysoglycolipids (e.g., GlcPSY and lysosulfatide), but not by SPC or N-acetyl PSY at concentrations up to 10 μM (data not shown).

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