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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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Generation and characterization of double transgenic FeCγ and single Fe65 transgenic mice. (A) Construction of AICD and Fe65 transgenes. The horizontal lines with arrows shows the location of transgene specific primers. (B) A PCR reaction on tail DNA isolated from three pups from Fe.27 line from three different litters (lanes 1–3) using Fe65 (left) or AICD primers (right) was performed together with primers for mouse Xist gene. Lanes denoted “+” contained DNA from the founder mouse (Fe.27). Note that none of the pups carries the transgene for AICD. (C and D) Western blot analysis of brain homogenates from two animals from double transgenic lines (FeCγ.12 and FeCγ.25), single Fe65 transgenic line (Fe.27), and nontransgenic littermate controls. Blots were probed with anti-myc 9E10 (C; top panel) or anti Fe65 antibody 3H6 (D; top panel), and visualized by ECL. The blots were stripped and reprobed with anti-tubulin DM1A antibody as an internal control (bottom panels). (E) Quantitative analysis of total Fe65 levels as detected by 3H6 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. Fe65 levels in FeCγ.12 and FeCγ.25 mice were significantly different from nontransgenic (nTg) animals (P < 0.0001), but not from Fe.27 mice (P = 0.04) by Bonferroni/Dunn test.
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fig1: Generation and characterization of double transgenic FeCγ and single Fe65 transgenic mice. (A) Construction of AICD and Fe65 transgenes. The horizontal lines with arrows shows the location of transgene specific primers. (B) A PCR reaction on tail DNA isolated from three pups from Fe.27 line from three different litters (lanes 1–3) using Fe65 (left) or AICD primers (right) was performed together with primers for mouse Xist gene. Lanes denoted “+” contained DNA from the founder mouse (Fe.27). Note that none of the pups carries the transgene for AICD. (C and D) Western blot analysis of brain homogenates from two animals from double transgenic lines (FeCγ.12 and FeCγ.25), single Fe65 transgenic line (Fe.27), and nontransgenic littermate controls. Blots were probed with anti-myc 9E10 (C; top panel) or anti Fe65 antibody 3H6 (D; top panel), and visualized by ECL. The blots were stripped and reprobed with anti-tubulin DM1A antibody as an internal control (bottom panels). (E) Quantitative analysis of total Fe65 levels as detected by 3H6 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. Fe65 levels in FeCγ.12 and FeCγ.25 mice were significantly different from nontransgenic (nTg) animals (P < 0.0001), but not from Fe.27 mice (P = 0.04) by Bonferroni/Dunn test.

Mentions: We cloned myc-tagged Fe65 or AICD in plasmid NN265 that contained intron and SV40 polyadenylation sequences (Abel et al., 1997). A fragment that contained the intron, the transgene open reading frame, and polyA signal was excised and cloned into MM403, downstream of the 8-kb CaMKIIα promoter (Fig. 1 A). We mixed AICD and Fe65 expressing plasmids in 1:1 proportion, and co-injected the linearized plasmids into oocytes of C57BL/6 mice. Injected oocytes were implanted in pseudopregnant C57BL/6 mice; by PCR on tail DNA, 9 out of 49 pups obtained were found to have incorporated both transgenes. All 9 founder mice were mated with C57BL/6 mice. Germline transmission was observed in five lines, of which four of the founder lines transmitted both transgenes to F1 pups (unpublished data). In the current study, we present data from two of these four independent lines (named FeCγ.12 and FeCγ.25). The fifth line, called Fe.27, did not transmit the AICD transgene to pups (Fig. 1 B), and thereby, fortuitously created a Fe65 single transgenic line. The expression levels of Fe65 transgene were determined by Western blot analysis. Total brain homogenates (40 μg protein each) from two animals from each transgenic line or two nontransgenic littermates was separated by SDS-PAGE on a 10% gel, transferred to a nitrocellulose membrane, and probed using anti-myc antibodies to detect the transgene or anti-Fe65 3H6 antibody to detect total Fe65 (endogenous + transgene). The myc-Fe65 signal was apparent in mice from all three transgenic lines, but was absent in nontransgenic littermates (Fig. 1 C, top panel). The total levels of Fe65 in the three transgenic lines (Fig. 1 D; top panel) were comparable, and were approximately twice as high as the nontransgenic control animals, when normalized for the levels of tubulin (Fig. 1 E). The Fe65 levels in Fe.27 mice were not significantly different from those in FeCγ.12 or FeCγ.25 mice (P = 0.04 by Bonferroni/Dunn test).


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

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

Generation and characterization of double transgenic FeCγ and single Fe65 transgenic mice. (A) Construction of AICD and Fe65 transgenes. The horizontal lines with arrows shows the location of transgene specific primers. (B) A PCR reaction on tail DNA isolated from three pups from Fe.27 line from three different litters (lanes 1–3) using Fe65 (left) or AICD primers (right) was performed together with primers for mouse Xist gene. Lanes denoted “+” contained DNA from the founder mouse (Fe.27). Note that none of the pups carries the transgene for AICD. (C and D) Western blot analysis of brain homogenates from two animals from double transgenic lines (FeCγ.12 and FeCγ.25), single Fe65 transgenic line (Fe.27), and nontransgenic littermate controls. Blots were probed with anti-myc 9E10 (C; top panel) or anti Fe65 antibody 3H6 (D; top panel), and visualized by ECL. The blots were stripped and reprobed with anti-tubulin DM1A antibody as an internal control (bottom panels). (E) Quantitative analysis of total Fe65 levels as detected by 3H6 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. Fe65 levels in FeCγ.12 and FeCγ.25 mice were significantly different from nontransgenic (nTg) animals (P < 0.0001), but not from Fe.27 mice (P = 0.04) by Bonferroni/Dunn test.
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fig1: Generation and characterization of double transgenic FeCγ and single Fe65 transgenic mice. (A) Construction of AICD and Fe65 transgenes. The horizontal lines with arrows shows the location of transgene specific primers. (B) A PCR reaction on tail DNA isolated from three pups from Fe.27 line from three different litters (lanes 1–3) using Fe65 (left) or AICD primers (right) was performed together with primers for mouse Xist gene. Lanes denoted “+” contained DNA from the founder mouse (Fe.27). Note that none of the pups carries the transgene for AICD. (C and D) Western blot analysis of brain homogenates from two animals from double transgenic lines (FeCγ.12 and FeCγ.25), single Fe65 transgenic line (Fe.27), and nontransgenic littermate controls. Blots were probed with anti-myc 9E10 (C; top panel) or anti Fe65 antibody 3H6 (D; top panel), and visualized by ECL. The blots were stripped and reprobed with anti-tubulin DM1A antibody as an internal control (bottom panels). (E) Quantitative analysis of total Fe65 levels as detected by 3H6 antibody. Protein levels were normalized to tubulin by reprobing the same blots after stripping. Quantification from three independent experiments. Values are the mean ± SEM; n = 6. Fe65 levels in FeCγ.12 and FeCγ.25 mice were significantly different from nontransgenic (nTg) animals (P < 0.0001), but not from Fe.27 mice (P = 0.04) by Bonferroni/Dunn test.
Mentions: We cloned myc-tagged Fe65 or AICD in plasmid NN265 that contained intron and SV40 polyadenylation sequences (Abel et al., 1997). A fragment that contained the intron, the transgene open reading frame, and polyA signal was excised and cloned into MM403, downstream of the 8-kb CaMKIIα promoter (Fig. 1 A). We mixed AICD and Fe65 expressing plasmids in 1:1 proportion, and co-injected the linearized plasmids into oocytes of C57BL/6 mice. Injected oocytes were implanted in pseudopregnant C57BL/6 mice; by PCR on tail DNA, 9 out of 49 pups obtained were found to have incorporated both transgenes. All 9 founder mice were mated with C57BL/6 mice. Germline transmission was observed in five lines, of which four of the founder lines transmitted both transgenes to F1 pups (unpublished data). In the current study, we present data from two of these four independent lines (named FeCγ.12 and FeCγ.25). The fifth line, called Fe.27, did not transmit the AICD transgene to pups (Fig. 1 B), and thereby, fortuitously created a Fe65 single transgenic line. The expression levels of Fe65 transgene were determined by Western blot analysis. Total brain homogenates (40 μg protein each) from two animals from each transgenic line or two nontransgenic littermates was separated by SDS-PAGE on a 10% gel, transferred to a nitrocellulose membrane, and probed using anti-myc antibodies to detect the transgene or anti-Fe65 3H6 antibody to detect total Fe65 (endogenous + transgene). The myc-Fe65 signal was apparent in mice from all three transgenic lines, but was absent in nontransgenic littermates (Fig. 1 C, top panel). The total levels of Fe65 in the three transgenic lines (Fig. 1 D; top panel) were comparable, and were approximately twice as high as the nontransgenic control animals, when normalized for the levels of tubulin (Fig. 1 E). The Fe65 levels in Fe.27 mice were not significantly different from those in FeCγ.12 or FeCγ.25 mice (P = 0.04 by Bonferroni/Dunn test).

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