Alzheimer’s disease is characterised by changes in the substance and structure of the brain that are caused by the depositing of protein plaques in the brain. Prof. Michael Przybylski and his team of researchers at the University of Constance have deciphered the structure of the site where mouse antibodies attach to the plaques, subsequently causing them to dissolve. These findings will now be used to identify the binding structure of a component of the human body’s immune defence that counteracts these deposits and to develop a selective vaccine.
Many years before the first symptoms can be seen, so-called amyloid plaques are formed and deposited in the brains of people who are affected by Alzheimer's. These plaques are the result of misfolded beta-amyloid (Aβ-) peptides, a form of organic protein compound produced by normal cells. "The more of these peptides that aggregate and the speedier the process, the quicker Alzheimer's develops," said Prof. Michael Przybylski. High-molecular aggregations and fibril structures affect the network of neurons, which lose their function. Some years ago, American scientists succeeded in producing antibodies in experimental mice. These antibodies bind to and dissolve beta-amyloid plaques, thereby improving the animals' memory and learning aptitude considerably.
The antibodies were able to recognise specific structures on the amyloid beta molecules on the basis of the key-lock principle and to subsequently remove the toxin. Following active vaccination with Aβ oligomers, the animals began to produce plaque-specific Aβ antibodies, which also prevented the development of new plaques. For the first time ever, Prof. Michael Przybylski and his team of 20 researchers were able to decipher the molecular recognition structures of these antibodies, i.e. epitopes, using biochemical analyses. They also discovered which epitopes were identified by the antibodies in the animal model.
The team of researchers used a method developed in their own laboratory. This method, known as proteolytic excision, was used in combination with mass spectroscopy to elucidate the structure of antibody epitopes. “We immobilised the plaque-specific antibodies on a surface and succeeded in binding the Aβ-containing material and degrading the non-binding areas using a selective protease,” explains Przybylski. These recognition epitopes were subsequently identified using mass spectrometry.The researchers initially found their findings somewhat paradoxical. “We were surprised to find that the Aβ material binds to a small peptide area of the Aβ antibody located a long way from the sequence stretch that leads to the aggregation of Aβ molecules,” said Prof. Michael Przybylski summarising their results. The clarification of the recognition epitopes forms the basis for developing both “highly specific diagnostics methods using peptide structures”, and lead structures for therapeutically active compounds.
Their work also involved the characterisation of genetic mutations since the cleavage of the precursor protein leads to large quantities of amyloid beta in a few genetic types of Alzheimer's that are associated with specific sequence alterations. The biochemical characterisation of the epitopes by the Constance researchers opens up many perspectives, particularly for the development of immunotherapeutic drugs for the treatment of Alzheimer's dementia. "We are now working on the identification of the epitope structure of the Aβ autoantibodies in Alzheimer's patients," said Prof. Michael Przybylski highlighting their plans to develop a selective and efficient vaccine, in which they will introduce multiple recognition epitope copies into suitable carrier molecules. If they are successful, the new antibodies will initially be tested for their efficacy and tolerability in mouse experiments.
The transfer of the vaccine to humans is a hugely challenging and important step in this process. Initial investigations on the vaccination using Aβ fragments in humans have been carried out in the past, but they had a severe outcome. "Although the tests have shown that Alzheimer's patients developed antibodies against amyloid and over time exhibited fewer disease symptoms than volunteers who did not develop any antibodies, the vaccinations nevertheless led to severe side effects such as meningitis and hence the trial was abandoned," said Prof. Michael Przybylski.
Besides the development of a therapeutic vaccine against Alzheimer's, Przybylski's group of researchers are also trying to gain insights into the development of a protective immunotherapeutic drug that could be applied as early as the first years of adolescence. "We still need to obtain many more insights into the physiological function of the Aβ autoantibodies," said the biochemist. The researchers assume that people who have sufficient quantities of Aβ autoantibodies are protected against Alzheimer's. According to Prof. Michael Przybylski, this opens up potential treatment strategies involving passive vaccination. In this case, the vaccination serum would consist of large quantities of the specific immunoglobulins directed against the typical deposits in the brain.
In future, the scientists will also look into why the Aβ autoantibodies do not lead to an autoimmune reaction. “We can only hypothesise that autoantibodies recognise the altered Aβ structure, which is then no longer seen as physiological, i.e. biological material,” said Przybylski. Therefore, there is good reason to believe that Aβ undergoes structural changes that are not yet known to the researchers.Recent studies have shown that Alzheimer plaques contain amyloid beta aggregates as well as other proteins, for example human cystatin C (HCC), a protease inhibitor. “The clarification of the Aβ binding eptitope revealed that cystatin C also interacts with an Aβ region that is responsible for aggregation,” said Przybylski. Therefore, the specific binding structures are regarded as potential lead structures for the development of vaccines. “We will only use the areas that are recognised by the epitope; this helps to prevent toxic effects,” said Przybylski.
Further information:University of ConstanceAnalytical Chemistry, Biopolymer ChemistryProf. Dr. Dr. Michael PrzybylskiTel.: +49 7531 882249Fax: +49 7531 883097E-mail: michael.przybylski(at)uni-konstanz.de