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P3‐197: TrkB isoforms differentially regulate amyloid precursor protein Swedish mutant glycosylation and processing
Author(s) -
Ansaloni Sara,
Leung Brian,
Gadaleta Mariana,
Saunders Aleister
Publication year - 2011
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2011.05.1638
Subject(s) - tropomyosin receptor kinase b , gene isoform , tropomyosin receptor kinase a , neurotrophin , microbiology and biotechnology , neurotrophic factors , receptor , biology , mutant , brain derived neurotrophic factor , alternative splicing , chemistry , biochemistry , gene
toxic species prone to aggregation. Moreover, using a variety of genetic and pharmacological manipulations we have found that close PS1 N-to C-termini proximity conformation correlates with the increased Ab42/40 ratio, whereas an “open” PS1 conformation (Nand C-termini are far apart) results in the decreased Ab42/40 ratio. Elevated Ab42/40 (total) ratio has also been reported in sporadic AD (sAD) and during normal aging. In addition, Ab imaging of cognitively normal aging adults, combined with postmortem studies, consistently report that w20-30% of individuals with no clinical symptoms of dementia show significant Ab deposition in the brain. This suggests that amyloid deposition precedes decline in cognition, and thus, may be the initiator in a cascade of events that lead to structural and functional disruption, and ultimately to age-related memory loss. Therefore, we hypothesize that, similar to genetic mutations in PS1, multiple age-related stress factors shift the PS1 conformation towards “close” PS1 NT-CT proximity, leading to a change in the PS1/g-secretase-substrate cleavage site in a way that favors cleavage of APP at the Aß42 position. We propose that this change in PS1 conformation precedes Aß plaque deposition, and is more pronounced in the vicinity to plaques. Methods: Fluorescence lifetime imaging microscopy, (FLIM), a Forster Resonance Energy Transfer (FRET)-basedmolecular imaging assay, was employed to study PS1 conformation in immunostained human andmouse brain tissue. The PS1Nand Ctermini were detected with epitope-specific antibodies labeled with Alexa488 and Cy3 as a donorand acceptor-fluorophores, respectively. PS1 conformation was analyzed in neurons within the CA1 hippocampal area. FRET efficiency (EFRET), as a measure of PS1 Nto C-termini proximity was calculated using the following equation: (EFRET%1⁄4 100*(t1/donor-t2/acceptor)/t1/donor), where t11⁄4decay lifetime of the Alexa 488 donor fluorophore alone; and t21⁄4 donor fluorophore lifetime in the presence of Cy3 acceptor fluorophore (FRET present). FRET efficiency increases, as the donor to acceptor fluorophore distance decreases. To monitor levels of Aß, PS1 and APP, Aß specific ELISA and western blot approaches were used. Human brain tissue samples of non-demented control individuals (n1⁄410, mean age 92.0 6 10.0 years), as well as AD (n1⁄410, mean age 86.5 6 9.9 years), and Fronto-temporal Dementia (FTD) (n1⁄45, mean age 71.76 24.8 years) patients were obtained from the Alzheimer Disease Research Center at Massachusetts General Hospital. To investigate the effects of aging on PS1 conformation Tg2576 transgenic mice expressing human sweAPP (Tg (HuAPP695.K670N-M671L)2576) and non-transgenic littermates (Charles River Laboratories) were used for longitudinal studies. Mice were divided into three groups based on pathological characteristics of the Tg2576 mouse model: young animals (1-4 months) with no amyloid deposition and no behavioral anomalies, young adults (7-9 months) with no obvious amyloid deposition but gradual onset of behavioral anomalies, and old animals (17-25 months) with pronounced amyloid deposits and behavioral anomalies. Results: To determine PS1 conformation EFRET was calculated in CA1 hippocampal neurons of sAD and FTD patients, or in age-matching non-dement.