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Tau phosphorylation by GSK-3beta promotes tangle-like filament morphology.

Rankin CA, Sun Q, Gamblin TC - Mol Neurodegener (2007)

Bottom Line: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology.Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved.This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA. crankin@ku.edu

ABSTRACT

Background: Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.

Results: We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3beta is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.

Conclusion: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

No MeSH data available.


Related in: MedlinePlus

Tau sites phosphorylated by GSK-3β in an in vitro reaction. Thirteen phosphorylation-site-specific antibodies were used to probe dot blots of GSK-3β phosphorylated tau. Panel A and B show two representative blots. Three repetitions of each phosphorylated tau concentration, ranging from 800 to 1.56 ng, were spotted on a blot, then probed with an anti-phosphorylation specific antibody (See Materials and Methods for details). To estimate non-specific background levels, each blot also included one spot of non-phosphorylated tau at each concentration. Following density analysis, the antibody titrations were plotted (Panels D and E) and 1/2 max concentrations were estimated. Anti-S422 antibody shown in Panel C, and its titration plot in Panel F, did not recognize phosphorylation and served as a negative control.
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Figure 2: Tau sites phosphorylated by GSK-3β in an in vitro reaction. Thirteen phosphorylation-site-specific antibodies were used to probe dot blots of GSK-3β phosphorylated tau. Panel A and B show two representative blots. Three repetitions of each phosphorylated tau concentration, ranging from 800 to 1.56 ng, were spotted on a blot, then probed with an anti-phosphorylation specific antibody (See Materials and Methods for details). To estimate non-specific background levels, each blot also included one spot of non-phosphorylated tau at each concentration. Following density analysis, the antibody titrations were plotted (Panels D and E) and 1/2 max concentrations were estimated. Anti-S422 antibody shown in Panel C, and its titration plot in Panel F, did not recognize phosphorylation and served as a negative control.

Mentions: We determined which tau sites were phosphorylated by utilizing a panel of phosphorylation specific antibodies. Thirteen dot blots containing concentrations of phosphorylated tau ranging from 1.56 to 800 ng per dot were probed with these antibodies. Representative dot blots are shown in Figure 2. Non-specific background binding to non-phosphorylated tau was subtracted and titration curves were plotted and analyzed (see Materials and Methods). The blot probed with anti-S199 showed very little non-specific antibody binding to non-phosphorylated tau (Figure 2A); anti-T217 showed slightly more non-specific binding (Figure 2B); and anti-S422 was the least specific antibody under these conditions (Figure 2C). The titration curves of most of the antibodies resembled that of anti-S199 (Figure 2D). These included anti-T205, -S214, -T231, -S262, -S356, -S400, -S404, and -S409. Anti-T212 and -S396 titration curves more closely resembled that of anti-T217 (Figure 2E). Only anti-tau S422 did not recognize a specific phosphorylation site (Figure 2C, F) and therefore, served as a negative control. The 1/2 max of most antibodies was in the range of 20–40 ng; only T212 differed, with a 1/2 max of roughly 12 ng. Our phosphorylation specific antibody data was in agreement with those sites recognized by the mass spectrometry data of Reynolds, et al. [37] and the combination of two dimensional phosphopeptide mapping and mass spectrometry used by Connell, et al. [38], except that we identified additional sites, T205, S214, S262, S356, and S409 that were not identified by mass spectrometry (Table 1). We did not have an antibody to the S235 site which was identified in both the Connell, et al. and Reynolds, et al. studies [37,38].


Tau phosphorylation by GSK-3beta promotes tangle-like filament morphology.

Rankin CA, Sun Q, Gamblin TC - Mol Neurodegener (2007)

Tau sites phosphorylated by GSK-3β in an in vitro reaction. Thirteen phosphorylation-site-specific antibodies were used to probe dot blots of GSK-3β phosphorylated tau. Panel A and B show two representative blots. Three repetitions of each phosphorylated tau concentration, ranging from 800 to 1.56 ng, were spotted on a blot, then probed with an anti-phosphorylation specific antibody (See Materials and Methods for details). To estimate non-specific background levels, each blot also included one spot of non-phosphorylated tau at each concentration. Following density analysis, the antibody titrations were plotted (Panels D and E) and 1/2 max concentrations were estimated. Anti-S422 antibody shown in Panel C, and its titration plot in Panel F, did not recognize phosphorylation and served as a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1936422&req=5

Figure 2: Tau sites phosphorylated by GSK-3β in an in vitro reaction. Thirteen phosphorylation-site-specific antibodies were used to probe dot blots of GSK-3β phosphorylated tau. Panel A and B show two representative blots. Three repetitions of each phosphorylated tau concentration, ranging from 800 to 1.56 ng, were spotted on a blot, then probed with an anti-phosphorylation specific antibody (See Materials and Methods for details). To estimate non-specific background levels, each blot also included one spot of non-phosphorylated tau at each concentration. Following density analysis, the antibody titrations were plotted (Panels D and E) and 1/2 max concentrations were estimated. Anti-S422 antibody shown in Panel C, and its titration plot in Panel F, did not recognize phosphorylation and served as a negative control.
Mentions: We determined which tau sites were phosphorylated by utilizing a panel of phosphorylation specific antibodies. Thirteen dot blots containing concentrations of phosphorylated tau ranging from 1.56 to 800 ng per dot were probed with these antibodies. Representative dot blots are shown in Figure 2. Non-specific background binding to non-phosphorylated tau was subtracted and titration curves were plotted and analyzed (see Materials and Methods). The blot probed with anti-S199 showed very little non-specific antibody binding to non-phosphorylated tau (Figure 2A); anti-T217 showed slightly more non-specific binding (Figure 2B); and anti-S422 was the least specific antibody under these conditions (Figure 2C). The titration curves of most of the antibodies resembled that of anti-S199 (Figure 2D). These included anti-T205, -S214, -T231, -S262, -S356, -S400, -S404, and -S409. Anti-T212 and -S396 titration curves more closely resembled that of anti-T217 (Figure 2E). Only anti-tau S422 did not recognize a specific phosphorylation site (Figure 2C, F) and therefore, served as a negative control. The 1/2 max of most antibodies was in the range of 20–40 ng; only T212 differed, with a 1/2 max of roughly 12 ng. Our phosphorylation specific antibody data was in agreement with those sites recognized by the mass spectrometry data of Reynolds, et al. [37] and the combination of two dimensional phosphopeptide mapping and mass spectrometry used by Connell, et al. [38], except that we identified additional sites, T205, S214, S262, S356, and S409 that were not identified by mass spectrometry (Table 1). We did not have an antibody to the S235 site which was identified in both the Connell, et al. and Reynolds, et al. studies [37,38].

Bottom Line: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology.Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved.This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA. crankin@ku.edu

ABSTRACT

Background: Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.

Results: We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3beta is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.

Conclusion: These results suggest that phosphorylation of tau by GSK-3beta promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

No MeSH data available.


Related in: MedlinePlus