A protein which aggravates Alzheimer's Disease

In mice, over-expression of ITPKB protein leads to exacerbation of Alzheimer's disease. Moreover, inhibition of ITPKB in cultured mouse neurons limits the production of amyloid peptides responsible for the formation of senile plaques.
Professor Stéphane Schurmans, director of the GIGA Functional Genetics Laboratory at the University of Liège, reveals the results of his work in a study published in Brain.

Genes hold the information required for making proteins, essential elements for living cells and hence life itself. Gene sequencing enables us to understand the composition of respective proteins, but not their function. However, proteins may play a multitude of different roles within the cell, because they ensure the vast majority of cell function. Proteins may thus play a role in structuring, catalysing and regulating DNA packaging, as well as in cell mobility and even gene expression.

To study the role of genes and the proteins which they encode, scientists use different techniques. 'For example, for enzymes, we can look at the substrates to which they bind, or to the product which results from the reaction that the enzyme catalyses, but also at the speed of this reaction or even try to determine where the enzyme is expressed. All these data enable us to obtain information on the role of this protein', explains Stéphane Schurmans, Director of the GIGA Laboratory of Functional Genetics at the University of Liège. 'But the ultimate aim is to be able to study the function of genes in vivo, i.e. at the level of the complete, living animal' he continues. And this is precisely the area which Stéphane Schurmans has been studying since 1992. He has genetically modified lab mice to obtain knock-out mice strains for target genes, i.e. mice in which a particular gene is inactivated. Around fifteen knock-out mice were created in the laboratory.

From the immune system to the nervous system

In 2003, Stéphane Schurmans and his team obtained the first knock-out mouse for the ITPKB gene (inositol 1, 4, 5 triphosphate 3-kinase B). 'We then discovered that these mice presented a particular phenotype because they did not have peripheral T lymphocytes , i.e. in the lymph nodes and the spleen', continues Professor Schurmans. Additional studies showed that other cells in the immune system, such as B lymphocytes, neutrophils, myeloid cells, and mast cells were also altered in ITPKB knock-out mice. 'These results show the importance of the role of the ITPKB enzyme in the haematopoietic system and the immune system', says the researcher. Thus, the ITPKB knock-out mice logically could be useful for the study of the ITPKB enzyme function within these biological systems.

In 2006, however, Schurmans was flabbergasted when he came across a Swedish study: 'biopsies carried out on patients affected by Alzheimer's Disease during this study showed the presence of a particularly high quantity of ITPKB messenger RNA in the sampled tissues'. Once he got over the shock, Schurmans was determined to find out more about the role of ITPKB in Alzheimer's Disease. He contacted Professor Jean-Pierre Brion at the Université Libre de Bruxelles, who specialised in Alzheimer's Disease. 'The idea was to combine our respective expertise, his in the area of this disease and mine in the area of the role of ITPKB and knock-out mice', explains Stéphane Schurmans.

Senile plaques and the ITPKB enzyme, a relationship of cause and effect?

Together, the two teams therefore decided to research the role of ITPKB in Alzheimer's Disease. 'The Swedish study showed a high level of ITPKB mRNA in patients but we wanted to check whether this coincided with a higher quantity of ITPKB protein in the cortex', explains Professor Schurmans. Samples obtained by Professor Jean-Pierre Brion enabled researchers to investigate this further, concluding that abnormally high quantities of the ITPKB protein were also present in the cortices of patients suffering from Alzheimer's Disease. In light of these very positive results, the scientists continued their work to identify where this protein is expressed. 'Sections taken from the brains of deceased patients who had Alzheimer's Disease show that ITPKB is largely localised in the neurons surrounding the amyloid plaques', reveals Schurmans. One of the characteristic signs of Alzheimer's Disease is the formation of amyloid plaques, also known as senile plaques. 'These are extracellular aggregates formed from amyloid peptides which precipitate and form plaques between the neurons. This is one of the causes of the disease', continues the Professor. Indeed, these senile plaques prevent the communication of information from one neuron to another. It should be noted that the neurons around the senile plaques are strongly suspected of secreting amyloid peptides' he adds.

Following these observations, the researchers decided to begin an in vitro study of cultured mouse neurons in which they over-expressed the ITPKB protein. The results did not disappoint the researchers: a large number of these neurons died and the cell culture supernanant provided evidence of highly increased production of amyloid peptide. 'However, these two things are precisely the particularities of Alzheimer's Disease', specifies Schurmans.

Over-expression does not trigger, but aggravates the disease

What is the effect of ITPKB over-expression in vivo? 'We have created mice which over-express the ITPKB enzyme specifically in the neurons of the hippocampus and the cerebral cortex, i.e. where changes are found in patients affected by Alzheimer's Disease', indicates the researcher. However, this experiment did not yield any particular observations. 'This means that the over-expression of ITPKB alone is insufficient to trigger Alzheimer's Disease and lead to the formation of senile plaques as well as neuron apoptosis', continues Schurmans. 'This enzyme probably does not play a major role in the disease, but may play a regulating role'. 

Far from being discouraged, the researchers over expressed the ITPKB protein in the brain of a genetically-modified mouse which develops an Alzheimer's Disease in less than 6 months. 'And bingo, we see that when we over-express ITPKB in the neurons of the hippocampus and the cortex of these Alzheimer mice, all the characteristics of the disorder are accentuated', reveals the Professor. A large quantity of ITPKB in the tissues involved in Alzheimer's Disease therefore aggravates the disease.

The role of the ITPKB enzyme is to transform inositol triphosphate (IP3) into inositol tetrakisphosphate (IP4). In this study, published in the journal Brain (1), the researchers also tested the effect of over-expression of a mutated form of the ITPKB enzyme, rendering it inactive. 'These experiments showed that, in this case, the disorder was not aggravated in the mouse', explains Schurmans. 'Importantly, this enzyme therefore catalyses IP4, the product of the reaction. It remains to be seen how IP4 accentuates the characteristics of Alzheimer's Disease.'

Towards a new inhibitor

In the near future, researchers are keen to investigate three new areas of research. The first consists of trying to understand why a minority of patients affected by Alzheimer's Disease do not exhibit high levels of ITPKB in the cortex and hippocampus. 'We also want to define the genetic or other factors which lead to the over-expression of ITPKB in the majority of patients', continues Schurmans. In the second area, research is aimed at determining whether the over-expression of this enzyme leads in humans, as it does in mice, to an aggravation of the disease. Finally, during preliminary experiments, researchers have shown that the use of an ITPKB inhibitor reduces the production of amyloid peptides by neurons in culture. Based on these results and recognising the potential of such a discovery, the major Swiss pharmaceutical company which develops this inhibitor has preferred to restrict the inhibitor to in-house use. 'We are currently working in partnership with researchers who are synthesising a new ITPKB inhibitor', says Professor Schurmans. 'If we can obtain a large quantity of this inhibitor, we will be able to see the effect which it has upon mouse models of Alzheimer's Disease'. At present, time should be set aside for largely investigative research, in order to assemble the pieces of the puzzle. Once these pieces fit, ITPKB may one day become a target for alleviating Alzheimer's Disease or slowing down its development.

(1) V. Stygelbout, K. Leroy, V. Pouillon, K. Ando, E. D’Amico,Y. Jia, H. R. Luo, C. Duyckaerts, C. Erneux, S. Schurmans and J.-P. Brion. Inositol trisphosphate 3-kinase B is increased in human Alzheimer brain and exacerbates mouse Alzheimer pathology. Brain. 2014 Jan 8.