Final Report Summary - EPIREGAD (Epigenetic regulation of Alzheimer's disease related genes)
The main aim of this project in the frame of Marie-Curie Intra-European Fellowship was to provide the fellow (Dr Eva Babusikova, Department of Biochemistry, Jessenius Faculty of Medicine, Comenius University in Bratislava, Martin, Slovakia) with the skills and expertise to carry out research aiming at validation of the epigenetic role of the amyloid precursor protein intracellular domain (AICD) in specific gene regulation, especially of genes of amyloid-degrading enzymes involved in pathogenesis of Alzheimer's disease (AD). The basic research performed during the project will lay a background for further validation of whether manipulation of these genes provides a viable therapeutic strategy in AD.
The work performed by the fellow during the 12 months of her fellowship involved both cellular models and animal studies. Using neuroblastoma SH-SY5Y cells over-expressing various isoforms of APP (APP695, APP751 and APP770) it was demonstrated that although the main amyloid-degrading enzyme neprilysin (NEP) was regulated by AICD in APP695 cells, other amyloid-degrading enzymes like endothelin-converting enzyme (ECE) and insulin-degrading enzyme (IDE) had different patterns of expression depending on APP isoforms with IDE being most likely AICD-independent. Since we have observed different cell proliferation rates in cells expressing various APP isoforms it was of special interest to check their effect on the levels of expression of a proapoptotic gene p53 and the levels of expression of histone deacetylases (HDACs) which affect gene expression. It was found that cells over-expressing APP isoforms had higher mRNA levels of p53 and reduced levels of expression of HDAC3 which might explain the changes observed in cell growth and proliferation and also in expression of the genes of interest.
Continuing this research we have investigated whether oxidative stress, which is one of the factors predisposing neuronal cells to death and promoting development of AD could modify regulation of genes of interest depending on APP metabolism and if this process was APP-isoform selective. It is well known that metabolism and processing of APP are affected by such conditions as hypoxia and oxidative stress and therefore we induced oxidative stress in cells by incubating them with hydrogen peroxide. The data obtained demonstrated that oxidative stress did not change expression of IDE in the cells over-expressing various APP isoforms. However, under oxidative stress, levels of ECE mRNA were decreased in all cell lines over-expressing APP isoforms compared to SH-SY5Y. On the contrary, levels of p53 mRNA were increased up to two times after oxidative stress but this effect was more pronounced in wild type cell lines compared with the cells over-expressing APP.
Because histone deacetylases are enzymes with a prominent role in epigenetic regulation during ageing and may participate in regulation of various neuronal genes, we have also analysed levels of expression of HDACs in different cell lines and the effect of oxidative stress on them. We have found that oxidative stress increased expression of HDAC1, HDAC3 and HDAC4 mRNA in all cell lines although this increase was less pronounced in the cells over-expressing APP695 compared to APP770. A parallel analysis of the effect of hypoxia on neuroblastoma NB7 cells has demonstrated that although hypoxia decreased levels of expression of NEP it had no pronounced effect on ECE and IDE expression. Taking into account that NEP expression depends on AICD presence on its promoter, it was further demonstrated that hypoxia results in decreased AICD promoter binding which correlated with reduced NEP mRNA levels and activity and the mechanisms of this regulation are currently under further investigation.
Since we have reported previously that an HDAC inhibitor valproic acid (VA) upregulates expression of NEP in wild type SH-SY5Y cells and in an animal model we have also analysed its effect in cells over-expressing various APP isoforms and subjected to oxidative stress. The results of these experiments demonstrated that treatment of the cells with VA resulted in an increased mRNA level of ECE and IDE but not of p53 in the wild type SH-SY5Y cells and cells over-expressing APP695 isoform although in APP-over-expressing cells the effect of VA on ECE and IDE was less pronounced. VA also inhibited expression of HDAC1 at the mRNA level in wild type cells but had no effect in the cells over-expressing APP695. Levels of HDAC3 and HDAC4 mRNA were also significantly decreased after treatment with VA although in APP695 cells expression of these enzymes was higher than in cells expressing other isoforms. Most importantly, VA had significant protective effect since it prevented the decrease of ECE expression and the increase of p53 levels caused by oxidative stress.
As previously we have reported that ischemia results in changes in proteins involved in APP metabolism we have extended our research to the analysis of the levels of expression of genes relevant to APP metabolism and pathogenesis of AD at the mRNA levels following ischemia and reperfusion in rats. The results of this study demonstrated that ischemia had a negative effect at the mRNA level of the main amyloid-degrading enzymes NEP and ECE. We have also found that levels of APP mRNA were increased in the hippocampus and in cortex of rats after ischemia. Expression of the enzyme involved in amyloidogenic processing of APP, namely BACE 1, at the mRNA level was also increased in rat brain after ischemic insult both in the hippocampus and cortex (up to 28 %) which supports our earlier observation that ischemia and hypoxia shifts APP processing in the brain structures involved in cognition towards amyloidogenic pathway. Reperfusion after ischemia did not significantly affect the level of BACE 1 at the protein level and mRNA levels.
Applying quantitative real-time PCR we have also found that levels of NEP expression were decreased by 25 - 28 % in brain hemispheres after ischemia compared to the control. These mRNA data were in agreement with the data on decreased NEP protein levels observed in rat brain after ischemia. Interestingly in the hippocampus, the decrease of NEP mRNA levels was even more dramatic (by 71 %) and after reperfusion it was still 51 % lower than in the control group of rats. Similarly to the protein levels, ECE mRNA expression in the hippocampus was also not changed after ischemia, however, ECE mRNA expression was decreased in the cortex (by 17 - 31 %) compared to the sham-operated control animals.
This study has also revealed that ischemia affects expression of histone deacetylases both in the brain hemispheres and in the hippocampus with the levels of HDAC3 mRNA being significantly increased compared to control. Taking into account our data on the role of HDAC3 in repression of NEP gene expression these findings explain reduced NEP expression in the ischemic brain. Altogether, the work performed in this part of the project provides further evidence that ischemia shifts APP processing towards its amyloidogenic pathway and also reduces expression of amyloid-degrading enzymes NEP and ECE in the cortex and hippocampus - the brain structures most affected in AD - and as such increase the risk of development of this pathology.
The work performed by Dr Babusikova during her fellowship in Leeds will be continued at her home institution where she will be focused on leadership of the research aimed at diagnosis and prevention of dementia and AD. She now has all the required skills and ambitions to achieve this and we are looking forward to a successful collabouration between the laboratories at the Universities of Leeds, UK and Martin, Slovakia.
The impact of this fellowship to the wider community will be both at the educational level since Dr Babusikova will transfer her knowledge obtained during her work in Leeds to the medical students she is currently teaching in Martin both at the undergraduate and postgraduate levels. Also the expertise she gained in Leeds will help the researchers in Martin to carry on work aimed at better understanding the molecular basis of pathogenesis of AD with the accent on mechanisms of clearance of toxic amyloid peptides via regulation of amyloid-degrading enzymes. This might have high therapeutic value especially with regard to the sporadic form of AD caused by ageing, ischemia, stroke and oxidative stress.
The work performed by the fellow during the 12 months of her fellowship involved both cellular models and animal studies. Using neuroblastoma SH-SY5Y cells over-expressing various isoforms of APP (APP695, APP751 and APP770) it was demonstrated that although the main amyloid-degrading enzyme neprilysin (NEP) was regulated by AICD in APP695 cells, other amyloid-degrading enzymes like endothelin-converting enzyme (ECE) and insulin-degrading enzyme (IDE) had different patterns of expression depending on APP isoforms with IDE being most likely AICD-independent. Since we have observed different cell proliferation rates in cells expressing various APP isoforms it was of special interest to check their effect on the levels of expression of a proapoptotic gene p53 and the levels of expression of histone deacetylases (HDACs) which affect gene expression. It was found that cells over-expressing APP isoforms had higher mRNA levels of p53 and reduced levels of expression of HDAC3 which might explain the changes observed in cell growth and proliferation and also in expression of the genes of interest.
Continuing this research we have investigated whether oxidative stress, which is one of the factors predisposing neuronal cells to death and promoting development of AD could modify regulation of genes of interest depending on APP metabolism and if this process was APP-isoform selective. It is well known that metabolism and processing of APP are affected by such conditions as hypoxia and oxidative stress and therefore we induced oxidative stress in cells by incubating them with hydrogen peroxide. The data obtained demonstrated that oxidative stress did not change expression of IDE in the cells over-expressing various APP isoforms. However, under oxidative stress, levels of ECE mRNA were decreased in all cell lines over-expressing APP isoforms compared to SH-SY5Y. On the contrary, levels of p53 mRNA were increased up to two times after oxidative stress but this effect was more pronounced in wild type cell lines compared with the cells over-expressing APP.
Because histone deacetylases are enzymes with a prominent role in epigenetic regulation during ageing and may participate in regulation of various neuronal genes, we have also analysed levels of expression of HDACs in different cell lines and the effect of oxidative stress on them. We have found that oxidative stress increased expression of HDAC1, HDAC3 and HDAC4 mRNA in all cell lines although this increase was less pronounced in the cells over-expressing APP695 compared to APP770. A parallel analysis of the effect of hypoxia on neuroblastoma NB7 cells has demonstrated that although hypoxia decreased levels of expression of NEP it had no pronounced effect on ECE and IDE expression. Taking into account that NEP expression depends on AICD presence on its promoter, it was further demonstrated that hypoxia results in decreased AICD promoter binding which correlated with reduced NEP mRNA levels and activity and the mechanisms of this regulation are currently under further investigation.
Since we have reported previously that an HDAC inhibitor valproic acid (VA) upregulates expression of NEP in wild type SH-SY5Y cells and in an animal model we have also analysed its effect in cells over-expressing various APP isoforms and subjected to oxidative stress. The results of these experiments demonstrated that treatment of the cells with VA resulted in an increased mRNA level of ECE and IDE but not of p53 in the wild type SH-SY5Y cells and cells over-expressing APP695 isoform although in APP-over-expressing cells the effect of VA on ECE and IDE was less pronounced. VA also inhibited expression of HDAC1 at the mRNA level in wild type cells but had no effect in the cells over-expressing APP695. Levels of HDAC3 and HDAC4 mRNA were also significantly decreased after treatment with VA although in APP695 cells expression of these enzymes was higher than in cells expressing other isoforms. Most importantly, VA had significant protective effect since it prevented the decrease of ECE expression and the increase of p53 levels caused by oxidative stress.
As previously we have reported that ischemia results in changes in proteins involved in APP metabolism we have extended our research to the analysis of the levels of expression of genes relevant to APP metabolism and pathogenesis of AD at the mRNA levels following ischemia and reperfusion in rats. The results of this study demonstrated that ischemia had a negative effect at the mRNA level of the main amyloid-degrading enzymes NEP and ECE. We have also found that levels of APP mRNA were increased in the hippocampus and in cortex of rats after ischemia. Expression of the enzyme involved in amyloidogenic processing of APP, namely BACE 1, at the mRNA level was also increased in rat brain after ischemic insult both in the hippocampus and cortex (up to 28 %) which supports our earlier observation that ischemia and hypoxia shifts APP processing in the brain structures involved in cognition towards amyloidogenic pathway. Reperfusion after ischemia did not significantly affect the level of BACE 1 at the protein level and mRNA levels.
Applying quantitative real-time PCR we have also found that levels of NEP expression were decreased by 25 - 28 % in brain hemispheres after ischemia compared to the control. These mRNA data were in agreement with the data on decreased NEP protein levels observed in rat brain after ischemia. Interestingly in the hippocampus, the decrease of NEP mRNA levels was even more dramatic (by 71 %) and after reperfusion it was still 51 % lower than in the control group of rats. Similarly to the protein levels, ECE mRNA expression in the hippocampus was also not changed after ischemia, however, ECE mRNA expression was decreased in the cortex (by 17 - 31 %) compared to the sham-operated control animals.
This study has also revealed that ischemia affects expression of histone deacetylases both in the brain hemispheres and in the hippocampus with the levels of HDAC3 mRNA being significantly increased compared to control. Taking into account our data on the role of HDAC3 in repression of NEP gene expression these findings explain reduced NEP expression in the ischemic brain. Altogether, the work performed in this part of the project provides further evidence that ischemia shifts APP processing towards its amyloidogenic pathway and also reduces expression of amyloid-degrading enzymes NEP and ECE in the cortex and hippocampus - the brain structures most affected in AD - and as such increase the risk of development of this pathology.
The work performed by Dr Babusikova during her fellowship in Leeds will be continued at her home institution where she will be focused on leadership of the research aimed at diagnosis and prevention of dementia and AD. She now has all the required skills and ambitions to achieve this and we are looking forward to a successful collabouration between the laboratories at the Universities of Leeds, UK and Martin, Slovakia.
The impact of this fellowship to the wider community will be both at the educational level since Dr Babusikova will transfer her knowledge obtained during her work in Leeds to the medical students she is currently teaching in Martin both at the undergraduate and postgraduate levels. Also the expertise she gained in Leeds will help the researchers in Martin to carry on work aimed at better understanding the molecular basis of pathogenesis of AD with the accent on mechanisms of clearance of toxic amyloid peptides via regulation of amyloid-degrading enzymes. This might have high therapeutic value especially with regard to the sporadic form of AD caused by ageing, ischemia, stroke and oxidative stress.