Effects of antibodies to phosphorylated and non-phosphorylated tau on in vitro tau phosphorylation at Serine-199: Preliminary report.
Phosphorylation of multiple amino acids on tau protein ("hyperphosphorylation") is required for the development of tau pathology in Alzheimer's disease. Administration of anti-tau antibodies to transgenic "tauopathy mice" has been shown to reduce their tau pathology but the mechanisms responsible are unclear. To examine the effects of anti-tau antibodies on tau phosphorylation, we used western blots to study the effects of three antibodies to phosphorylated tau (pTau), namely anti-pTau S199, T231, and S396, and three antibodies to non-phosphorylated tau on in vitro phosphorylation of recombinant human tau-441 at S199. Inclusion of an anti-pTau T231 antibody in the phosphorylation reaction reduced the intensity of monomeric pTau S199 in western blots of denaturing gels, but the other antibodies had no apparent effects on this process. Surprisingly, including all three anti-phospho-tau antibodies in the reaction did not reduce the intensity of the monomer band, possibly due to steric hindrance between the antibodies.


CONCLUSIONS
These preliminary findings suggest that anti-tau antibodies may have minimal direct effects on tau phosphorylation. Limitations of using western blots to examine the effects of anti-tau antibodies on this process were found to include between-experiment variability in pTau band densities and poor resolution of high molecular weight pTau oligomers. The presence of bands representing immunoglobulins as well as pTau may also complicate interpretation of the western blots. Further studies are indicated to examine the effects of anti-pTau antibodies on phosphorylation of other tau amino acids in addition to S199.
Department of Neurology, Beaumont Research Institute, Beaumont Health System, 3601 West Thirteen Mile Road, Royal Oak, MI 48073, USA. Electronic address: DLoeffler@beaumont.edu.
David A
DA
Loeffler
Department of Neurology, Beaumont Research Institute, Beaumont Health System, 3601 West Thirteen Mile Road, Royal Oak, MI 48073, USA.
Lynnae M
LM
Smith
Department of Neurology, Beaumont Research Institute, Beaumont Health System, 3601 West Thirteen Mile Road, Royal Oak, MI 48073, USA.
Andrea C
AC
Klaver
Department of Chemistry, Oakland University, Rochester, MI 48309, USA.
Sanela
S
Martić
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western / Q000379:methods; D066298:In Vitro Techniques; D008822:Mice, Transgenic; D010766:Phosphorylation / Q000276:immunology; D012694:Serine / Q000276:immunology*; D024801:Tauopathies / Q000276:immunology*; D016875:tau Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D016875:tau Proteins; D012694:Serine
Alzheimer's disease; Antibodies; Hyperphosphorylation; Tau; Western blots