The PSP Association

Lead researcher Dr Diane Hanger
Co-researchers Selina Wray (PhD student), Dr Tamas Revesz and Dr Janice Holton

Institution MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry.

Duration 3 years
Start October 2004 End September 2007

Grant £33,300 (part funding for a three year PhD studentship; co-funded by the Medical Research Council).

Aim of Research

Progressive supranuclear palsy (PSP) is characterised neuropathologically by tangles in the brain containing aggregates of the cytoskeletal protein, tau, in neurons and glia. Increased phosphorylation of PSP tau is the most likely cause of this deposition, which results in nerve cell death. The aims of this project were to:

• Enrich insoluble PSP tau from frozen human brain tissue affected by PSP.
• Purify PSP tau from the enriched sample to homogeneity.
• Use the purified material to identify phosphorylation sites on PSP tau by mass spectrometric analysis of phosphopeptides.
• Compare the phosphorylation state of PSP tau with that of insoluble tau purified from Alzheimer brain with the aim of determining protein kinases that may be involved in tau deposition in neurodegenerative disease.

About the research
The first stage of this project was to screen multiple regions of different PSP brains stored in the brain bank to determine which brains and which regions contained consistently the largest amounts of insoluble tau. A region was selected and a larger amount of this material was used for further biochemical analysis. Insoluble PSP tau was enriched and purified from the selected region using a sequential process of differential centrifugation, heat-stability and chromatography. Soluble tau from control brain and insoluble tau from Alzheimer's disease brain were also purified for comparison with PSP tau. The purified tau was then analysed by mass spectrometry to determine the full complement of phosphorylation sites on PSP tau. The information derived from this research provides important information and may help to identify responsible protein kinases.

Findings
We found that the pre-motor cortex of PSP brain produced the greatest yield of insoluble tau. However, the deposited tau in PSP brain appeared to have different biochemical characteristics from that of Alzheimer tau and therefore a new method had to be developed to enrich for and purify PSP tau. Using the new method we were able to purify PSP tau in sufficient yield for mass spectrometric analysis from pre-motor cortex although, as we had suspected previously, we found that the yield of insoluble tau from PSP brain was significantly lower than that obtained using similar procedures for Alzheimer brain tissue. Mass spectrometry identified eight phosphorylation sites on PSP tau, three of which have not been reported previously on PSP tau and one of these was not detected on Alzheimer tau by direct means. These results therefore suggest that there may be a difference in the degree of phosphorylation at this latter site between the two related disorders of Alzheimer's disease and PSP. Furthermore, we identified a major cleavage fragment of tau in PSP brain tissue that may be specific to disorders in which particular forms of tau are deposited (the so-called 4R tauopathies, including PSP, corticobasal degeneration, and certain dementias). This latter finding occurred towards the end of this period of funding and led to an application for further funding from the PSP Association.

What does the outcome of this research mean for people with PSP?
The cause of PSP is not known and therefore any information that builds on our knowledge of factors involved in disease progression is important to discover the mechanisms involved in the development of PSP. This is the first report of direct identification of tau phosphorylation sites on material extracted from PSP brain. The major difference between Alzheimer and PSP tau appears to be primarily in the degree of phosphorylation that occurs as well as possible quantitative differences at specific sites. The suggestion from this work is that therapies targeted at reducing tau phosphorylation in Alzheimer's disease could have potential benefit in PSP. However, there may be subtle but important differences in phosphorylation between the two disorders.

Publications arising from the work

Tau Phosphorylation Article - 'Trends in Molecular Medicine', Mar 09

Wray, S., Saxton, M., Anderton, B.H., and Hanger, D.P. (2008). Direct analysis of tau from PSP brain identifies new phosphorylation sites and a major fragment of N-terminally cleaved tau containing four microtubule-binding repeats. J. Neurochem, in press. PMID: 18315566.

Reynolds, C.H., Garwood, C.J., Wray, S., Price, C., Kellie, S., Perera, T., Zvelebil, M., Yang, A., Sheppard, P.W., Varndell, I.M., Hanger, D.P. and Anderton, B.H. (2008). Phosphorylation regulates tau interactions with SH3 domains of phosphatidylinositol-3-kinase, phospholipase c1, grb2 and src-family kinases. J. Biol. Chem. 283, 18177-18186.

Hanger, D.P., Byers, H.L., Wray, S., Leung, K.-Y., Saxton, M.J., Seereeram, A., Reynolds, C.H., Ward, M.A., and Anderton, B.H. (2007). Novel phosphorylation sites in tau from Alzheimer brain support a role for casein kinase 1 in disease pathogenesis. J. Biol. Chem., 282, 23645-54.

National/international presentations given on this work
Wray, S., Anderton, B.H. and Hanger, D.P. Potential toxicity of Tau cleavage products in progressive supranuclear palsy. 11th International conference on Alzheimer's disease and related disorders, Chicago, USA. July 25-31, 2008.

Wray S., Anderton B.H. and Hanger D.P. Tau phosphorylation and cleavage in progressive supranuclear palsy. 6th forum of European Neuroscience, Geneva, Switzerland, July 12-16, 2008.

Hanger D. P., Wray S., Seereeram A., Noble W and Anderton B. H. Protein phosphorylation and tau kinases implicated in the tauopathies. MRC Showcase: Breakthroughs in neuroscience and mental health, Bristol, UK, February, 2008

Ward, M., Fernandez-Ocana, M., Schofield, E., Wray, S., Hanger, D., Byers, H., and Anderton, B.H. Multi-site phosphorylation assays for tau protein and their relevance to Alzheimer's disease and other neurological disorders. European biomarkers summit and proteomics Europe conference. 2007.

Wray, S., Revesz, T., Holton, J.L., Anderton, B.H., Hanger, D.P. (2006) Enrichment of insoluble tau from progressive supranuclear palsy brain tissue. 10th international conference on Alzheimer's disease and related disorders, Madrid, Spain. Alzheimer's & Dementia, 2, S466.

An article featuring this research entitled Untangling tau protein' was included in the King's College London, Institute of Psychiatry Research Report, 2008-9. Page 9.

Selina Wray presented this work at the 2008 MRC Centre for Neurodegeneration Research Open Day which involves communicating research to members of the public in order to raise awareness of PSP and related disorders.

Weblinks
King's College London, staff
MRC Centre for Neurodegeneration Research

How has the research you conducted influenced your career and research direction?
The results of this research have led to several successful applications for funding to continue this work including:
The PSP Association: salary support for Selina Wray for one year
Psychiatry Research Trust: funds for generating a specific antibody
Biomedical Research Centre: salary support for Selina Wray for 3 months.