Differential effects of familial Alzheimer’s disease-causing mutations on amyloid precursor protein (APP) trafficking, proteolytic conversion, and synaptogenic activity

The amyloid precursor protein (APP) is a key player in Alzheimer`s disease (AD) and the precursor of the Aβ peptide, which is generated by consecutive cleavages of β- and γ-secretases. Familial Alzheimer’s disease (FAD) describes a hereditary subgroup of AD that represents a low percentage of AD cases with an early onset of the disease. Different APP FAD mutations are thought to have qualitatively different effects on its proteolytic conversion. However, few studies have explored the pathogenic and putative physiological differences in more detail. Here, we compared different FAD mutations, located at the β- (Swedish), α- (Flemish, Arctic, Iowa) or γ-secretase (Iberian) cleavage sites. We examined heterologous expression of APP WT and FAD mutants in non-neuronal cells and their impact on presynaptic differentiation in contacting axons of co-cultured neurons. To decipher the underlying molecular mechanism, we tested the subcellular localization, the endocytosis rate and the proteolytic processing in detail by immunoprecipitation–mass spectrometry. Interestingly, we found that only the Iberian mutation showed altered synaptogenic function. Furthermore, the APP Iowa mutant shows significantly decreased α-secretase processing which is in line with our results that APP carrying the Iowa mutation was significantly increased in early endosomes. However, most interestingly, immunoprecipitation–mass spectrometry analysis revealed that the amino acid substitutions of APP FAD mutants have a decisive impact on their processing reflected in altered Aβ profiles. Importantly, N-terminally truncated Aβ peptides starting at position 5 were detected preferentially for APP Flemish, Arctic, and Iowa mutants containing amino acid substitutions around the α-secretase cleavage site. The strongest change in the ratio of Aβ40/Aβ42 was observed for the Iberian mutation while APP Swedish showed a substantial increase in Aβ1–17 peptides. Together, our data indicate that familial AD mutations located at the α-, β-, and γ-secretase cleavage sites show considerable differences in the underlying pathogenic mechanisms

Mild Reductive Cleavage of α-Aminoethers

l,2,3,4-Tetrahydro-6,7-dimethoxy-2-methylisoquinoline (1) is converted by ethyl chloroformate (ECF)/NaBH3CN to 2-[ß-(N-ethoxycarbonyl-N-methyl)aminoethyl]-4,5-dimethoxytoluene (4) via the quaternary urethane 2, The same procedure leads from laudanosine (5) to the dibenzyl derivative 9. The reaction with ECF/NaBH3CN followed by LiAlH4 reduction is a versatile approach to Emde degradation products avoiding strongly basic conditions and elevated temperature. Cleavage reactions of other α-amino ethers, e.g. thebaine (18), and N-demethylation reactions of the tetrahydroisoquinolines 1 and 10 with ECF are reported. l,2,3,4-Tetrahydro-6,7-dimethoxy-2-methylisochinolin (1) wird mit Chlorameisensäureethylester (ECF)/NaBH3CN über das quartäre Urethan 2 zum 2-[(ß-N-Ethoxycarbonyl-N-methyl)-aminoethyl]- 4,5-dimethoxytoluol (4) umgesetzt. - Dieses Verfahren führt von Laudanosin (5) zum Dibenzyl-Derivat 9. - Die ECF/NaBH3CN-Reaktion, kombiniert mit der LiAlH4-Reduktion der tert. Urethane, ist eine Alternative zum Emde-Abbau und vermeidet stark basische Bedingungen und erhöhte Temp. Die Spaltung weiterer α-Aminoether, u.a. Thebain (18), und N-Demethylierungen der Tetrahydroisochinoline 1 und 10 werden beschrieben