Amylin and pramlintide modulate γ-secretase activity and APP processing in lipid rafts

Abstract

The misfolded amyloid-β (Aβ) peptide is generated in higher amounts in Alzheimer’s disease (AD), making this peptide a clinical hallmark for AD. A major site to produce Aβ is the lipid rafts, which are integral part of the cell membrane and enriched with cholesterol and sphingolipids. These rafts contain proteins and enzymes that are involved in the production of Aβ, in addition to other proteins such as synaptic markers and transport proteins. To isolate these rafts, in the first project, I developed and optimized a density-based separation method utilizing discontinuous sucrose gradient with ultracentrifugation. Different factors could control the efficiency of rafts fractionation, such as type of detergent, ultracentrifugation time and speed, rotor type, and cells or tissue type, thus, these factors were initially optimized to isolate lipid rafts containing proteins of interest in one fraction. Consequent to the optimization, membrane rafts were successfully isolated and localized in one fraction, which contained proteins related to my second project objective including proteins related to Aβ production, synaptic markers and gangliosides. Several studies have reported the protective effect of amylin pramlintide against AD. On the other hand, other studies demonstrated amylin rather worsen AD pathology. In either case, the mechanisms by which amylin improved or deteriorated AD pathology are not well investigated. In addition, studies evaluated the effect of pramlintide against AD are limited. Thus, the purpose of this work was to investigate the effect of amylin and pramlintide on Aβ-related pathology in TgSwDI mice as a model for AD, and to investigate the predisposing mechanism for the observed effect. Therefore, in the second project, we explored the effect of amylin and pramlintide on Aβ-related pathology in TgSwDI mice. After chronic intraperitoneal treatment for 30 days with amylin or pramlintide, brains were collected and evaluated. Findings from immunostaining and ELISA demonstrated increased accumulation of Aβ in mice brains treated with amylin or pramlintide when compared to vehicle treated mice. To explain the observed effect, findings from total brain homogenate didn’t provide a clear justification for Aβ increase, thus lipid rafts were used for further studies. Results from lipid rafts analyses demonstrated that both peptides increased Aβ burden by increasing the level of amyloid precursor protein (APP) and γ-secretase, an Aβ producing enzyme, in lipid rafts. One major pathway that control the localization of APP and γ-secretase activity in lipid rafts, and increased Aβ production and aggregation is the increased level of gangliosides, such as GM1 and GM2 gangliosides. Pramlintide increased GM1 and GM2 levels in lipid rafts and total brain homogenate, respectively. As expected, increased Aβ burden in mice brains was associated with synaptic loss, apoptosis, microglial activation, and increased Aβ deposition on brain microvessels. In conclusion, findings from this work demonstrated amylin and pramlintide increased Aβ levels and related pathology in TgSwDI mice brains, implying that the increased amylin level or the therapeutic use of pramlintide might increase the risk of AD.