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  • Final Report Summary - γ-SECRETASE AGING (Study of the aging-related changes of the gamma-secretase complex and evaluation to what extent those contribute to amyloid accumulation in sporadic Alzheimer’s disease)

Final Report Summary - γ-SECRETASE AGING (Study of the aging-related changes of the gamma-secretase complex and evaluation to what extent those contribute to amyloid accumulation in sporadic Alzheimer’s disease)

Despite the striking fact that Alzheimer’s Disease (AD) is an age-dependent pathology, we lack still a satisfactory explanation at the molecular level linking the age-associated changes that occur in neurons and the triggering of the disease (reviewed in 1). AD is the most common cause of human dementia and is histopathologically characterized by the presence of cerebral senile plaques composed of Aβ peptide. Mostly based on studies of families with inherited AD (FAD), it is assumed that abnormal Aβ generation is the initial trigger of the disease process (the amyloid hypothesis) (2,3). FAD is characterized by small increases in the ratio of Aβ42 versus Aβ40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients (4). Little is known however whether aging, the major risk factor for sporadic AD, affects Aβ generation as well. We hypothesize that the enzymatic activity of the γ-secretase complex, which is the ultimate responsible for Aβ generation, could be altered during aging and in sporadic AD, mimicking the changes found in FAD. Thus, the project entitled “Study of the aging-related changes of the γ-secretase complex and evaluation to what extent those contribute to amyloid accumulation in sporadic Alzheimer’s disease” contains three lines of research: 1-The study of changes of the γ-secretase activity in sporadic AD, 2- The effect in vitro and in vivo of oxidative stress on the γ-secretase activity and 3- the role of lipid composition of the membrane on the activity of the complex.

For the first line of research, we compared the enzymatic activity of the γ-secretase complex from the cerebral frontal cortex of 20 controls and 20 sporadic AD patients, obtained from the London Brain Bank and the “Banc de Teixits Neurologics” of The Hospital Clinic of Barcelona. We then analyzed the activity of the solubilized fraction, paying special attention to the Aβ42/Aβ40 ratio, since it is found elevated in AD. In opposition to what we expected, we detected a slight decrease of the Aβ42/Aβ40 ratio in AD patients due to a diminished production of Aβ42 peptide. This could be the consequence of a compensatory mechanism aimed to decrease the levels of the more aggregative Aβ42 peptide once the patient has already accumulated too much Aβ. However a large fraction of presenilin-1 (PS1), the catalytic activity of the complex, was resistant to solubilization in the AD samples with the conditions needed to perform the assay and its activity remained unstudied. Taking into account this pool of PS1, its levels were found increased in the cerebral cortex of patients with AD. Since aging is associated to increased oxidative damage to proteins, next we analyzed the presence of oxidative modifications on γ-secretase by western blot and using specific antibodies. PS1 was found to be highly nitrotyrosinated in the brain of AD patients, indicative of high levels of nitrosative stress. In summary, we found that the portion of γ-secretase activity studied changes in brains with advanced AD towards a decrease production of Aβ42. However we do not know if aging couples with switched cleavage of APP towards an increased Aβ42/Aβ40 ratio, which is compensated in advanced stages of the disorder. Finally the total PS1 levels were found elevated in sporadic AD and largely nitrotyrosinated.

For the second line of research, we studied the changes occurring in the γ-secretase complex in rat hippocampal neurons during aging. To address this point, we established the conditions to allow primary rat neurons to age in vitro. Once seeded in the culture dish, hippocampal neurons undergo a whole series of morphological and functional maturation processes reflecting those of their counterparts in vivo. Because of the results obtained during the first line of research, we focused on the effect of nitrotyrosination on γ-secretase activity. We found that aging of neurons in vitro increases the nitrotyrosination of γ-secretase, triggering an AD-like change in the conformation and activity of the complex, leading to an elevated Aβ42/Aβ40 ratio. We confirmed similar changes in a Sod2 knockout mouse model, an in vivo model for oxidative stress. These findings, added to the augmented nitrotyrosination of PS1 in the brains of individuals affected with sporadic AD, add clinical value to the mechanistic association depicted in these lines. This study provides a molecular explanation to the important question how ageing predisposes to AD. More concretely, our findings show that the age-associated increase of nitrosative stress drives γ-secretase towards a switch of the Aβ profile favouring the Aβ42 species. This effect on the complex mimics the characteristic treat of FAD-mutations and sets the γ-secretase as a target not only for inherited but also for late-onset AD. The work has been successfully published in EMBO Molecular Medicine (5).

Finally, we proceeded to the third line of research to study how the lipidic composition of the plasma membrane can modulate the activity of γ-secretase. Several studies have focused on the effect of detergent-resistant membranes enriched in cholesterol and sphingomyeline on the generation of Aβ. However little attention has been paid to the processing of APP in detergent-soluble microdomains enriched in phospholipids. For this part of the project, we collaborated with the laboratory of Carlos Dotti. Since the membrane levels of phosphatidylethanolamine (PE) are altered in AD, but not those of phosphatidylinositol neither phosphatidylcholine (6,7), we decided to focus on the effect of this lipid on the activity of γ-secretase. In mammal cells the reduction of PE levels by siRNA silencing of the enzyme involved in its synthesis, ethanolamine kinase, reduces the γ-secretase-dependent Aβ generation. In Drosophila Melanogaster the genetic reduction of the PE levels decreases the γ-secretase-dependant APP cleavage. This part of the project opens the possibility of a therapeutic intervention devoted to decrease the generation of Aβ by modulating the PE levels. The study was published in Aging Cell (8).


1- Kern A, Behl C (2009) The unsolved relationship of brain aging and late-onset Alzheimer disease. Biochim Biophys Acta. 1790:1124-32
2- Wakabayashi T, De Strooper B (2008) Presenilins: members of the gamma-secretase quartets, but part-time soloists too. Physiology (Bethesda). 23:194-204
3- Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297:2209
4- Chávez-Gutiérrez L, Bammens L, Benilova I, Vandersteen A, Benurwar M, Borgers M, Lismont S, Zhou L, Van Cleynenbreugel S, Esselmann H, Wiltfang J, Serneels L, Karran E, Gijsen H, Schymkowitz J, Rousseau F, Broersen K, De Strooper B (2012) The mechanism of γ-Secretase dysfunction in familial Alzheimer disease. EMBO J. 31:2261-74
5- Guix FX, Wahle T, Vennekens K, Snellinx A, Chávez-Gutiérrez L, Ill-Raga G, Ramos-Fernandez E, Guardia-Laguarta C, Lleó A, Arimon M, Berezovska O, Muñoz FJ, Dotti CG, De Strooper B (2012) Modification of γ-secretase by nitrosative stress links neuronal ageing to sporadic Alzheimer's disease. EMBO Mol Med. 4:660-73
6- Wells K, Farooqui AA, Liss L, Horrocks LA (1995) Neural membrane phospholipids in Alzheimer disease. Neurochem. Res. 20:1329–1333
7- Prasad MR, Lovell MA, Yatin M, Dhillon H, Markesbery WR (1998) Regional membrane phospholipid alterations in Alzheimer’s disease. Neurochem. Res. 23:81–88
8- Nesic I, Guix FX, Vennekens K, Michaki V, Van Veldhoven PP, Feiguin F, De Strooper B, Dotti CG, Wahle T (2012) Alterations in phosphatidylethanolamine levels affect the generation of Aβ. Aging Cell. 11:63-72

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