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Identification of microtubule-associated protein tau isoforms in Alzheimer's paired helical filaments
L McLaughlin1, F P Zemlan, G E Dean
1Alzheimer's Research Center, Department of Psychiatry, University of Cincinnati College of Medicine, OH 45267, USA.
Abstract:
AD66 proteins derived from sodium dodecylsulfate (SDS) insoluble paired helical filaments (PHF) were isolated from Alzheimer's brain using a purification procedure developed previously in this laboratory, and characterized by immunologic and chemical cleavage methods. AD66 proteins were immunoreactive with antibodies that recognize the amino terminal, tubulin-binding, and carboxy terminal domains of microtubule-associated protein tau indicating the presence of the entire tau sequence in AD66 proteins. These proteins were reactive with antibody 423 that binds to PHF but not human adult tau. Immunologic and chemical cleavage studies indicated that only two of the six tau isoforms were present in these proteins. AD66 proteins were comprised of tau proteins containing only three tubulin binding domains with either a 29 amino acid insert or no amino terminal insert. For comparative purposes, SDS soluble PHF-tau (A68 proteins) was purified from Alzheimer's brains and normal adult tau purified from control brains. Antibody Alz-50 was immunoreactive with PHF-tau or normal tau regardless of alkaline phosphatase treatment while immunoreactivity was only observed with dephosphorylated AD66 proteins. A second phosphorylated epitope on AD66 proteins but not PHF-tau or normal tau proteins was demonstrated with antibody PHF9. These data suggest that AD66 proteins represent a more phosphorylated form of tau than PHF-tau or normal tau proteins. Two-dimensional gel electrophoresis demonstrated that AD66 proteins have higher apparent molecular weights and lower pI values than normal tau, differences possibly due to the greater phosphorylation observed in these proteins.
Insights
AD66 proteins from Alzheimer's brain tau are more phosphorylated than standard PHF-tau. These proteins contain the full tau sequence but lack specific tau isoforms and exhibit altered biochemical properties.
Area of Science:
- Neuroscience
- Biochemistry
- Alzheimer's Disease Research
Background:
- Paired helical filaments (PHF) composed of hyperphosphorylated tau protein are a hallmark of Alzheimer's disease (AD).
- Understanding the specific tau species present in AD pathology is crucial for developing targeted therapies.
Purpose of the Study:
- To characterize AD66 proteins, isolated from SDS-insoluble PHF in Alzheimer's brains.
- To compare the biochemical and immunological properties of AD66 proteins with SDS-soluble PHF-tau (A68) and normal adult tau.
Main Methods:
- Purification of AD66 proteins from Alzheimer's brain PHF.
- Immunological characterization using antibodies against tau domains and specific epitopes.
- Chemical cleavage analysis.
- Comparative analysis with purified A68 proteins and normal tau.
- Two-dimensional gel electrophoresis.
Main Results:
- AD66 proteins contain the entire tau sequence, including tubulin-binding domains, with specific inserts.
- AD66 proteins react with antibody 423 (binds PHF) but not with normal adult tau.
- Only two of six tau isoforms are present in AD66 proteins.
- AD66 proteins show differential immunoreactivity with antibodies Alz-50 and PHF9 compared to PHF-tau and normal tau, suggesting higher phosphorylation.
- Two-dimensional gel electrophoresis indicates higher molecular weight and lower pI for AD66 proteins, consistent with increased phosphorylation.
Conclusions:
- AD66 proteins represent a distinct, highly phosphorylated tau species found in Alzheimer's disease.
- These findings suggest that tau undergoes specific modifications and isoform processing within PHF.
- The unique phosphorylation pattern of AD66 proteins may contribute to neurofibrillary tangle formation and neuronal dysfunction in Alzheimer's disease.