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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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Analysis of mRNA transcript levels by real-time PCR. Total RNA from at least three animals was isolated, subjected to reverse transcriptase, and real-time PCR was performed using BioRad iCycler and primers for KAI1 gene (A) or GSK-3β gene (B). Mouse β-actin primers were used as an internal control. Data are expressed as levels of KAI1 or GSK-3β transcripts relative to β-actin. Values are the mean ± SEM; n = 6. P = 0.08 for KAI1 transcript levels in FeCγ.12 against nontransgenic or Fe.27 mice by Fisher's PLSD test.
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fig5: Analysis of mRNA transcript levels by real-time PCR. Total RNA from at least three animals was isolated, subjected to reverse transcriptase, and real-time PCR was performed using BioRad iCycler and primers for KAI1 gene (A) or GSK-3β gene (B). Mouse β-actin primers were used as an internal control. Data are expressed as levels of KAI1 or GSK-3β transcripts relative to β-actin. Values are the mean ± SEM; n = 6. P = 0.08 for KAI1 transcript levels in FeCγ.12 against nontransgenic or Fe.27 mice by Fisher's PLSD test.

Mentions: In contrast to the in vitro observations that AICD causes an increase in the GSK-3β protein levels in tissue culture cells, we did not observe an increase in the GSK-3β protein levels in the transgenic mice (Fig. 4, A and C). To examine this discrepancy in detail, we measured the mRNA levels in brain extracts by real-time PCR. Total RNA was extracted from brain tissue, subjected to reverse transcriptase reaction, and real-time PCR was performed. We used primers for GSK-3β and KAI1, and normalized the values by using β-actin. FeCγ.12 mice showed a 1.8-fold increase in KAI1 message when compared with nontransgenic or Fe.27 mice (Fig. 5 A). By contrast, we detected no change in GSK-3β transcripts when compared with nontransgenic or Fe.27 mice (Fig. 5 B). These observations suggest that GSK-3β activation in FeCγ transgenic mice is not due to increased transcription or translation of GSK-3β gene. Because GSK-3β kinase activity is regulated largely at the posttranslational level by upstream kinases and phosphatases (Jope and Johnson, 2004), it is possible that AICD activates GSK-3β by modulating upstream regulators. However, because the transgenes are expressed only in certain neuronal cells, we cannot rule out the possibility that a slight increase in GSK-3β mRNA or protein was undetected in whole brain extracts.


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

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

Analysis of mRNA transcript levels by real-time PCR. Total RNA from at least three animals was isolated, subjected to reverse transcriptase, and real-time PCR was performed using BioRad iCycler and primers for KAI1 gene (A) or GSK-3β gene (B). Mouse β-actin primers were used as an internal control. Data are expressed as levels of KAI1 or GSK-3β transcripts relative to β-actin. Values are the mean ± SEM; n = 6. P = 0.08 for KAI1 transcript levels in FeCγ.12 against nontransgenic or Fe.27 mice by Fisher's PLSD test.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171208&req=5

fig5: Analysis of mRNA transcript levels by real-time PCR. Total RNA from at least three animals was isolated, subjected to reverse transcriptase, and real-time PCR was performed using BioRad iCycler and primers for KAI1 gene (A) or GSK-3β gene (B). Mouse β-actin primers were used as an internal control. Data are expressed as levels of KAI1 or GSK-3β transcripts relative to β-actin. Values are the mean ± SEM; n = 6. P = 0.08 for KAI1 transcript levels in FeCγ.12 against nontransgenic or Fe.27 mice by Fisher's PLSD test.
Mentions: In contrast to the in vitro observations that AICD causes an increase in the GSK-3β protein levels in tissue culture cells, we did not observe an increase in the GSK-3β protein levels in the transgenic mice (Fig. 4, A and C). To examine this discrepancy in detail, we measured the mRNA levels in brain extracts by real-time PCR. Total RNA was extracted from brain tissue, subjected to reverse transcriptase reaction, and real-time PCR was performed. We used primers for GSK-3β and KAI1, and normalized the values by using β-actin. FeCγ.12 mice showed a 1.8-fold increase in KAI1 message when compared with nontransgenic or Fe.27 mice (Fig. 5 A). By contrast, we detected no change in GSK-3β transcripts when compared with nontransgenic or Fe.27 mice (Fig. 5 B). These observations suggest that GSK-3β activation in FeCγ transgenic mice is not due to increased transcription or translation of GSK-3β gene. Because GSK-3β kinase activity is regulated largely at the posttranslational level by upstream kinases and phosphatases (Jope and Johnson, 2004), it is possible that AICD activates GSK-3β by modulating upstream regulators. However, because the transgenes are expressed only in certain neuronal cells, we cannot rule out the possibility that a slight increase in GSK-3β mRNA or protein was undetected in whole brain extracts.

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