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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 18, 2002 as doi:10.1096/fj.02-0434fje. |
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,2
,2
* Department of Physiology and
Department of Anatomy, School of Medicine, Ehime University, Ehime 791-0295, Japan;
Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan;
Department of Anatomy, Ajou University School of Medicine, Suwon 442–749, Korea;
|| Department of Anatomy, Kobe University School of Medicine, Kobe 650-0017, Japan; and
Division of Neurology, Duke University Medical Center, Durham, North Carolina, USA.
3Correspondence: Department of Physiology, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-0295, Japan. E-mail: mitsuda{at}m.ehime-u.ac.jp
SPECIFIC AIMS
The association of apolipoprotein E (ApoE) genotypes with Alzheimer’s disease, reelin’s association with ApoE receptors, ApoE receptor association with intracellular signaling machinery, and increased levels of phosphorylated tau suggest a novel signal transduction pathway where ApoE receptor-mediated signaling follows a series of steps that targets an increase in tau protein phosphorylation. By measuring the distribution of phosphorylated tau, tau kinases, and phosphatases, we examined molecular mechanisms underlying the accumulation of phosphorylated tau proteins in reeler and yotari mice in the presence or absence of ApoE.
PRINCIPAL FINDINGS
1. Levels of site-specific tau phosphorylation increase in reelin (Reln)-deficient mice as ApoE protein levels decrease
To investigate the relationship between levels of ApoE receptor ligands and tau phosphorylation, we generated mice that lacked Reln and expressed normal levels of mouse ApoE protein (Reln-/- ApoE+/+), lacked Reln and expressed half of the normal level of ApoE protein (Reln-/- ApoE+/-), and lacked Reln and ApoE proteins (Reln-/- ApoE-/-). Lysates of cerebral cortices from these and wild-type mice (Reln+/+ ApoE+/+) were Western blotted and probed with antibodies to detect total tau protein levels (5E2), levels of tau protein not phosphorylated at a specific site (Tau-1), and levels of tau protein phosphorylated at specific sites relative to an internal control of ß-tubulin (Fig. 1
A, B). Phosphorylation at specific sites was measured by immunostaining with anti-phospho-tau antibodies AT270, 44–752, 44–758, 44–760, and 44–764, which react with phosphorylated residues at Thr-172, Ser-387, Ser-395, Ser-400, and Ser-413.
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The highest levels of tau phosphorylation at Thr-172, Ser-387, and Ser-395 were seen in mice lacking Reln and ApoE (Reln-/- ApoE-/-), followed by Reln-/- ApoE+/- mice, and Reln-/- ApoE+/+, whereas little immunostaining was observed in wild-type mice (Reln+/+ ApoE+/+). The anti-phospho-tau antibodies 44–760 and 44–764, which react with phosphorylated residues at Ser-400 and Ser-413, consistently immunostained tau proteins isolated from each of the four different genotypes as did the phosphorylation independent antibody 5E2 directed to the region between residues 142 and 166 (Fig. 1A
). Although the total amount of protein (ß-tubulin stain) and the total amount of tau protein (5E2 stain) loaded per lane were equivalent, levels of phosphorylation at specific sites on tau in Reln-deficient mice were significantly increased as the amount of ApoE proteins decreased (Fig. 1A, B
).
2. Levels of site-specific tau phosphorylation are increased in disabled-1 (Dab1)-deficient mice independent of ApoE protein
Disabled-1 is a cytosolic adaptor protein that specifically binds protein motifs such as those found on the cytoplasmic tails of integral transmembrane ApoE receptors. Assembly of a Reln/ApoE receptor/Dab1 complex (RAD) may initiate a signal transduction cascade that inhibits tau phosphorylation whereas disruption of the RAD complex appears to increase tau phosphorylation. We tested this hypothesis by generating mice that lacked Dab1 and expressed normal levels of mouse ApoE protein (Dab1-/- ApoE+/+) and lacked Dab1 and ApoE proteins (Dab1-/- ApoE-/-). Western blots of cerebral cortical lysates of these and wild-type mice (Reln+/+ ApoE+/+) were probed with the same anti-tau antibodies as described above (Fig. 1C, D
). Levels of phosphorylated tau proteins isolated from Dab1-/- ApoE+/+ and Dab1-/- ApoE-/- mice were higher than those isolated from wild-type mice (Dab1+/+ ApoE+/+). These levels of residue-specific phosphorylation did not differ between Dab1-/- ApoE+/+ and Dab1-/- ApoE-/- mice except for antibody 44–764 to Ser-413, indicating that the observed increase in phosphorylation was generally independent of ApoE levels in these mice (Fig. 1C
).
3. Glycogen synthase kinase-3ß (GSK-3ß) activity and protein levels are increased as ApoE protein levels decrease in Reln-deficient mice, but are independent of ApoE in Dab1-deficient mice
Using in vitro kinase assays, we found that GSK-3ß activity levels in wild-type brains were significantly lower than in Reln-deficient and Dab1-deficient mice. When combined with changes in ApoE levels, we found that GSK-3ß activity levels were highest in Reln-/- ApoE-/- mice, significantly lower in Reln-/- ApoE+/- mice, and still lower in Reln-/- ApoE+/+ mice, even though each of these mice demonstrated kinase activity levels significantly higher than in wild-type. The highest level of GSK-3ß activity was observed in lysates of mice lacking Reln and ApoE and was comparable to its activity in lysates of mice lacking Dab1 or lacking Dab1 and ApoE. There were no significant differences between these three highest groups (Fig. 2
A). Western blots show that immunoreactive GSK-3ß was lower in wild-type lysates than in lysates from mice lacking ApoE, Reln or Dab1 (Fig. 2B
). Similar to the activity levels, the concentration of immunoreactive GSK-3ß was lowest in wild-type brain lysates and gradually increased in Reln-deficient, Reln-deficient and ApoE-reduced, and Reln- and ApoE-deficient lysates (Fig. 2C
).
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CONCLUSIONS
Our study demonstrates that ApoE protein limits protein kinase activity and levels of phosphorylated tau protein in Reln-deficient mice, but not in the brains of Dab1-deficient mice. These data support a novel role for the ApoE receptor and its ligands, ApoE and Reln, in regulating tau protein phosphorylation through a novel signal transduction cascade. Specific biochemical assays of brain lysates demonstrated that the activity of GSK-3ß increased significantly in Reln-deficient mice and that its activity further increased to a significantly higher degree in mice lacking Reln and ApoE. This pattern of increased GSK-3ß activities almost exactly matched the progressive and significantly increased levels of tau protein phosphorylation on Thr-172, Ser-387, and Ser-395 residues, suggesting that GSK-3ß caused this increase in site-specific tau phosphorylation. GSK-3ß activity increased in Dab1-deficient mice to the same levels as seen in Dab1- and ApoE-deficient mice, a pattern that matches well with the levels of phosphorylated tau residues and again suggests that GSK-3ß may be responsible for this increase in site-specific tau phosphorylation.
In conclusion, our studies provide genetic and biochemical evidence that ligands modulate tau phosphorylation through a novel signal transduction pathway. These ligands bind to ApoE receptors to initiate a signaling cascade that suppresses kinase activities, thereby reducing the level of tau protein phosphorylation (see Fig. 3
). When the pathway functions normally, ApoE and/or Reln proteins are ligands that bind to one or more ApoE receptors, signaling adaptor proteins associated with the cytoplasmic tails of the receptors (such as Dab1) to activate an unknown chain of events that suppresses the activity of GSK-3ß kinase. Dab1 also activates an unknown chain of events that suppresses the conversion of p35 to p25, thereby inhibiting production of this constitutive activator and suppressing CDK-5 activity. Reducing the activity of GSK-3ß, CDK-5, or both inhibits tau phosphorylation and thus protects neurons from the damaging effects of hyperphosphorylated tau and from accumulation of neurofibrillary tangles.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0434fje; to cite this article, use FASEB J. (December 18, 2002) 10.1096/fj.02-0434fje 
2 N.O. and Y.-D. L. equally contributed to this work.
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