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Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.

Ryan KA, Pimplikar SW - J. Cell Biol. (2005)

Bottom Line: APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm.In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined.Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3beta (GSK-3beta) and phosphorylation of CRMP2 protein, a GSK-3beta substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

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The AICD transgenic mice show activated GSK-3 levels. (A) Higher levels of activated form of GSK-3α and -3β in AICD transgenic mice. Immunoblot analysis of brain cytosol from animals from indicated lines was probed with anti-GSK antibody (pY279/216) that specifically recognizes the activated forms of GSK-3α and -3β enzymes (top panel). Note that mice from both AICD transgenic lines show higher levels of activated GSK-3α and -3β (lanes 3–6) as compared with control (con; lanes 1 and 2) or Fe.27 mice (lanes 7 and 8). Total GSK-3β protein levels are not changed (bottom panel). (B) Quantitative analysis of phospho–GSK-3β levels in transgenic and control mice. Quantification of GSK-3α levels gave similar results (not depicted). Protein levels were normalized to tubulin by reprobing the same blots after stripping. This experiment was repeated twice, and was performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (C and D) AICD transgenic mice show a dramatic reduction in the levels of inhibited form of GSK-3β, as detected by pS9–GSK-3β antibody (top panel). The total GSK-3β levels were not changed (bottom panel). Quantitative analysis of data in shown in (D). The protein levels were normalized to tubulin. This experiment was repeated twice and performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. **, P < 0.001 against nontransgenic or Fe.27 mice. (E) Activated ERK1 and ERK2 levels are not altered significantly in transgenic mice as detected by pERK1/2 antibodies.
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fig4: The AICD transgenic mice show activated GSK-3 levels. (A) Higher levels of activated form of GSK-3α and -3β in AICD transgenic mice. Immunoblot analysis of brain cytosol from animals from indicated lines was probed with anti-GSK antibody (pY279/216) that specifically recognizes the activated forms of GSK-3α and -3β enzymes (top panel). Note that mice from both AICD transgenic lines show higher levels of activated GSK-3α and -3β (lanes 3–6) as compared with control (con; lanes 1 and 2) or Fe.27 mice (lanes 7 and 8). Total GSK-3β protein levels are not changed (bottom panel). (B) Quantitative analysis of phospho–GSK-3β levels in transgenic and control mice. Quantification of GSK-3α levels gave similar results (not depicted). Protein levels were normalized to tubulin by reprobing the same blots after stripping. This experiment was repeated twice, and was performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (C and D) AICD transgenic mice show a dramatic reduction in the levels of inhibited form of GSK-3β, as detected by pS9–GSK-3β antibody (top panel). The total GSK-3β levels were not changed (bottom panel). Quantitative analysis of data in shown in (D). The protein levels were normalized to tubulin. This experiment was repeated twice and performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. **, P < 0.001 against nontransgenic or Fe.27 mice. (E) Activated ERK1 and ERK2 levels are not altered significantly in transgenic mice as detected by pERK1/2 antibodies.

Mentions: Glycogen synthase kinase (GSK)–3β is a proline-directed Ser/Thr kinase that is implicated in AD pathogenesis (Jope and Johnson, 2004). In vitro observations suggest that ectopic expression of AICD results in a significant increase in the mRNA and protein levels of GSK-3β (Kim et al., 2003; Von Rotz et al., 2004). To examine whether AICD activates GSK-3β in vivo, we determined the status of GSK-3 by using antibodies that recognize the activated or inhibited forms of the enzyme. Phosphorylation of GSK-3β at Y216 stimulates its kinase activity (active form), whereas phosphorylation at S9 potently represses (inactive form) the kinase activity (Jope and Johnson, 2004). Brain cytosolic fractions were immunoblotted with antibody anti-pY216/279 that recognizes the phospho-Y216 on GSK-3β and the equivalent Y279 residue on GSK-3α. Fig. 4 A shows that activated forms of GSK-3α and -3β were elevated in FeCγ.12 and FeCγ.25 mice (top panel, lanes 3–6) when compared with nontransgenic controls (lanes 1 and 2) or Fe.27 mice (lanes 7 and 8). Densitometric analysis revealed (Fig. 4 B) an ∼1.7-fold increase in pGSK-3β levels in FeCγ animals (GSK-3α showed similar changes). We also analyzed the total GSK-3β levels by stripping the blots and reprobing with antibodies that recognize GSK-3β and observed no significant changes in the protein levels in transgenic mice compared with the control animals (Fig. 4 A, bottom panel).


Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain.

Ryan KA, Pimplikar SW - J. Cell Biol. (2005)

The AICD transgenic mice show activated GSK-3 levels. (A) Higher levels of activated form of GSK-3α and -3β in AICD transgenic mice. Immunoblot analysis of brain cytosol from animals from indicated lines was probed with anti-GSK antibody (pY279/216) that specifically recognizes the activated forms of GSK-3α and -3β enzymes (top panel). Note that mice from both AICD transgenic lines show higher levels of activated GSK-3α and -3β (lanes 3–6) as compared with control (con; lanes 1 and 2) or Fe.27 mice (lanes 7 and 8). Total GSK-3β protein levels are not changed (bottom panel). (B) Quantitative analysis of phospho–GSK-3β levels in transgenic and control mice. Quantification of GSK-3α levels gave similar results (not depicted). Protein levels were normalized to tubulin by reprobing the same blots after stripping. This experiment was repeated twice, and was performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (C and D) AICD transgenic mice show a dramatic reduction in the levels of inhibited form of GSK-3β, as detected by pS9–GSK-3β antibody (top panel). The total GSK-3β levels were not changed (bottom panel). Quantitative analysis of data in shown in (D). The protein levels were normalized to tubulin. This experiment was repeated twice and performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. **, P < 0.001 against nontransgenic or Fe.27 mice. (E) Activated ERK1 and ERK2 levels are not altered significantly in transgenic mice as detected by pERK1/2 antibodies.
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fig4: The AICD transgenic mice show activated GSK-3 levels. (A) Higher levels of activated form of GSK-3α and -3β in AICD transgenic mice. Immunoblot analysis of brain cytosol from animals from indicated lines was probed with anti-GSK antibody (pY279/216) that specifically recognizes the activated forms of GSK-3α and -3β enzymes (top panel). Note that mice from both AICD transgenic lines show higher levels of activated GSK-3α and -3β (lanes 3–6) as compared with control (con; lanes 1 and 2) or Fe.27 mice (lanes 7 and 8). Total GSK-3β protein levels are not changed (bottom panel). (B) Quantitative analysis of phospho–GSK-3β levels in transgenic and control mice. Quantification of GSK-3α levels gave similar results (not depicted). Protein levels were normalized to tubulin by reprobing the same blots after stripping. This experiment was repeated twice, and was performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. *, P < 0.05 against nontransgenic (nTg) or Fe.27 mice by Fisher's PLSD test. (C and D) AICD transgenic mice show a dramatic reduction in the levels of inhibited form of GSK-3β, as detected by pS9–GSK-3β antibody (top panel). The total GSK-3β levels were not changed (bottom panel). Quantitative analysis of data in shown in (D). The protein levels were normalized to tubulin. This experiment was repeated twice and performed on animals from an independent FeCγ.22 line. Values are the mean ± SEM; n = 6. **, P < 0.001 against nontransgenic or Fe.27 mice. (E) Activated ERK1 and ERK2 levels are not altered significantly in transgenic mice as detected by pERK1/2 antibodies.
Mentions: Glycogen synthase kinase (GSK)–3β is a proline-directed Ser/Thr kinase that is implicated in AD pathogenesis (Jope and Johnson, 2004). In vitro observations suggest that ectopic expression of AICD results in a significant increase in the mRNA and protein levels of GSK-3β (Kim et al., 2003; Von Rotz et al., 2004). To examine whether AICD activates GSK-3β in vivo, we determined the status of GSK-3 by using antibodies that recognize the activated or inhibited forms of the enzyme. Phosphorylation of GSK-3β at Y216 stimulates its kinase activity (active form), whereas phosphorylation at S9 potently represses (inactive form) the kinase activity (Jope and Johnson, 2004). Brain cytosolic fractions were immunoblotted with antibody anti-pY216/279 that recognizes the phospho-Y216 on GSK-3β and the equivalent Y279 residue on GSK-3α. Fig. 4 A shows that activated forms of GSK-3α and -3β were elevated in FeCγ.12 and FeCγ.25 mice (top panel, lanes 3–6) when compared with nontransgenic controls (lanes 1 and 2) or Fe.27 mice (lanes 7 and 8). Densitometric analysis revealed (Fig. 4 B) an ∼1.7-fold increase in pGSK-3β levels in FeCγ animals (GSK-3α showed similar changes). We also analyzed the total GSK-3β levels by stripping the blots and reprobing with antibodies that recognize GSK-3β and observed no significant changes in the protein levels in transgenic mice compared with the control animals (Fig. 4 A, bottom panel).

Bottom Line: APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm.In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined.Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Cell Biology Program, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Amyloid precursor protein (APP), implicated in Alzheimer's disease, is a trans-membrane protein of undetermined function. APP is cleaved by gamma-secretase that releases the APP intracellular domain (AICD) in the cytoplasm. In vitro studies have implicated AICD in cell signaling and transcriptional regulation, but its biologic relevance has been uncertain and its in vivo function has not been examined. To investigate its functional role, we generated AICD transgenic mice, and found that AICD causes significant biologic changes in vivo. AICD transgenic mice show activation of glycogen synthase kinase-3beta (GSK-3beta) and phosphorylation of CRMP2 protein, a GSK-3beta substrate that plays a crucial role in Semaphorin3a-mediated axonal guidance. Our data suggest that AICD is biologically relevant, causes significant alterations in cell signaling, and may play a role in axonal elongation or pathfinding.

Show MeSH
Related in: MedlinePlus