Benefits and detrimental effects of sequence variants of Amyloid-β: towards the use of small peptides for aggregate dissolution therapy in dementia


General information
The project is co-financed by the European Economic Area Financial Mechanism and the Norwegian Financial Mechanism 2009-2014 Programme LV05 ‘Research and Scholarships’.

Project no. NFI/R/2014/023
Implementation period: 1 May  2015 – 30 April 2017
Project promoter: Latvian Institute of Organic Synthesis  (IOS)
Partner 1: University of Latvia (LU)
Partner 2: University of Oslo (UiO)
Project total cost:  485 999,00 EUR
Allocation from Norway Grants: 400 949,00 EUR (82.5%)
National co-financing: 48 600,00  EUR (10%)
Self co-financing:  36 450,00 (7.5%)

The programme  LV05 “Research and Scholarships” is administered by the Ministry of Education and Science of the Republic of Latvia in collaboration with the State Education Development Agency.
Project  summary
The overall goal of this project is to develop the new strategy for the treatment of the Alzheimer disease (AD) and to enhance research-based  knowledge in Latvia through a long term collaboration of IOS, LU and UiO. The Latvian research groups at IOS will provide their expertise in structural analyses to start an iterative set of experiments. The UiO will provide knowledge and expertise in the field of disease models and analyses. The LU partners will have access to world-wide unique mouse models, and are skilled in the performance of behavioural and cognitive tests.
The outcome from this Latvian-Norwegian research cooperation project will be the identification of neuroprotective properties of short N-terminal peptides of Aβ for their propensity to remove toxic Aβ peptide deposits and improve cognitive functions which may introduce new options in AD therapy.

              • Project progress from 01.01.2017. till 30.04.2017.

                15N labeled Aβ40A2V spectra have been assign the 15N and 1H chemical shifts of the peptide. Based on these assignments, we have confirmed previously Thioflavin T measurements with NMR spectroscopy. We have also used solid state NMR to study fibril morphology of Aβ40 A2V and Aβ42 WT. Although, so far, these experiments have not yet been successful due to polydisperse samples and optimization is ongoing. We have also made constructs with a solubility tag from the nursery web spider Euprosthenops australis to prevent Aβ from aggregating and have expressed and purified Aβ42WT and Aβ42A2V fusion proteins. These fusion proteins were loaded into Alzet pumps (model 2006 and 2004) and implanted into 50-days-old and 75-days-old  male APP/PS1 +/0 mice, respectively. We have also cloned, express and started to purify 1-15 truncations of WT and A2V variants of the Aβ peptide. These truncated peptides will also be loaded into Alzet pumps and implanted after the purification process.The project has so far generated 12 publications in high ranked journals such as Acta Neuropathologica Communications and Nature Communication. In total, we have cloned and expressed up to 15 different clones of the Aβ peptide. We are currently investigating the properties of a new solubility tag from the golden orb-web spider Nephila clavipes fused to the Aβ peptide to facilitate protein expression.Published on 09.05.2017.

              • Project progress from 01.11.2016. till 31.12.2016.

                15N labeled Aβ40A2V have been produced with our new construct and 2D 15N HSQC, 3D-15N edited NOESY and TOCSY spectra have been acquired to assign the 15N and 1H chemical shifts of the peptide. This was necessary to characterize the peptide dynamics and probe the conformation of high molecular, NMR invisible species. We have also been working on optimizing ITC experimental conditions to determine the Aβ40 peptide affinity for Cu2+ ions. Solid-state NMR 15N HSQC spectrum has been acquired on the 13C and 15N labelled Aβ42 fibers.
                Published on 16.01.2017.

            • Project progress from 01.08.2016. till 31.10.2016.

              Our improved Aβ construct have now been optimized in terms of production and purification and we now get extraordinary yields in both LB and M9 media. This new construct has been used as a template for new clones such as hAβ40, hAβ42, hAβ2-40, hAβ2-42, hAβ40A2V, hAβ42A2V etc. Primers for producing truncated variants of Aβ, i.e. Aβ1-15 and Aβ1-15A2V have been ordered. Further cloning has been performed on the solubility tag to increase binding to the IMAC column. The 13C and 15N labelled Aβ42 sample has been purified and incubated at 37oC to fibrillate.
              Published on 10.11.2016.

            • Participation of principal investigator Henrik Biverstal at the 20th Swedish Conference on Protein Structure and Function

            • Project progress from 01.05.2016. till 31.07.2016.

              To current date project team has cloned and started production of Aβ40, Aβ42, Aβ40A2V, Aβ42A2V and two truncated versions of the wild type (Aβ2-40 and Aβ2-42). The new, improved construct of Aβ42 have been expressed in isotope enriched (13C-glucose and 15N-NH4Cl) and will be purified, fibrillated and used for structural elucidation with solid state NMR. Moreover, various solution NMR techniques have been used to characterize the interaction between Aβ40 and hexapeptides.
              Published on 01.08.2016.

            • EEA and Norway Grants contribution to the development of research and mobility
              (video including summary of the project)

            • Project progress from 31.01.2016. till  30.04.2016.

              LIOS have made two constructs based on Aβ(A2V)1-6 with two different cell penetrating peptides tags to assist passage over the blood brain barrier, TAT-Aβ(A2V)1-6 and AntP-Aβ(A2V)1-6. A new method for overexpression of Aβ and variants thereof has also been established. All these new constructs are currently being investigated.
              To reveal the positive effects of different mutated amyloid beta peptides, in vivo experiments (surgical implantation of Alzet pumps carrying different amyloid beta peptides) are taking place in the laboratory of the Norwegian partner Prof. Jens Pahnke.
              LU researchers are investigating the short sequence peptides using in vivo models (by implementing behavioral and cognitive tests) and ex vivo methods (immunohistochemical staining and western blotting).
              Staff mobility from LU to the University of Oslo has been done in order to obtain high quality skills in animal database acquisition, surgery with Alzet pumps on mice and method validation.
              Published on 10.05.2016.

            • Project progress from 01.11.2015. till  31.01.2016.

              The cloning of hAβ42 to mAβ42 and combinations between the two isoforms has been finished. Three mutants has been designed (hAβ42R5G, hAβ42Y10F, hAβ42H13R) and also full mouse Aβ42 as well (R5G, Y10F and H13R in respect to hAβ). All four constructs have been produced and purified in accordance with earlier protocols. All four constructs have similar aggregation kinetics as hAβ42WT, hAβ42A2V and hAβ42A2T.
              To deliver these short peptides into the model organism laboratory mice’s brain, we are using specific pumps (Alzet pumps). These Alzet pumps contains the peptides in their “body”, and when they are implanted under the skin in the back of the animal they start to pump the peptides (via a tube and a cannula which is fixed to the skull) into the brain. Since we have problems to properly fix the cannula into the mice skull in the preliminary pilot brain infusion experiments, and because of the inappropriate fixing the brain could be harmed, a new method to fix the cannula to solve this problem has been developed in the laboratory of the Norwegian partner prof. Jens Pahnke. The application of this new method makes the experiments easier to manage and to repeatable and also helps to reduce the number of the used animals by reducing the probability of producing damaged brains.
              Published on 10.02.2016.

              • Participation of principal investigator Henrik Biverstal at the international ISMAR 2015 Conference

              • Project progress in IOS from 01.08.2015. till 31.10.2015.

                The production, purification and analysis of amyloid-beta peptide variants with different sequences has been done.
                The following peptides were obtained for further studies:
                1) amyloid-beta peptide 1-40;
                2) amyloid-beta peptide 1-42;
                3) amyloid-beta peptide 1-6 mutant A2V;
                4) amyloid-beta peptide 1-6 mutant A2T.
                Initial interaction studies between amyloid-beta peptide 1-42 and amyloid-beta peptide 1-6 mutant A2V were performed.
                Published on 09.11.2015.

              • Project progress in IOS from 01.05.2015. till 31.07.2015.

                The design and preparation of amyloid-beta peptide expression constructs with different sequences has been made :
                1) amyloid-beta peptide 1-40;
                2) amyloid-beta peptide 1-42;
                3) amyloid-beta peptide 1-42 mutant A2V;
                4) amyloid-beta peptide 1-42 mutant A2T.
                Work has begun on the production and purification of these peptides.
                Published on 10.08.2015.