Alzheimer disease : studies on Abeta and gamma-secretase in human brain

Sammanfattning: Alzheimer disease (AD) is a devastating neurodegenerative disorder and the most prevalent form of dementia. One hallmark of the disease is the extracellular deposition of amyloid beta-peptide (Abeta) into senile plaques in the brain. Biochemical and genetic studies reveal Abeta as a key player in AD pathogenesis. The most common forms of Abeta are 40 (Abeta40) or 42 (Abeta42) residues long. Abeta40 is produced at higher levels than Abeta42; while Abeta42 is more hydrophobic, prone to aggregate, and form the toxic species. Thus, the length of the hydrophobic C-terminus of Abeta is very important for oligomerization and neurotoxicity. Abeta is generated through sequential processing of the amyloid beta- precursor protein (APP) by the enzymes beta- and gamma-secretases. The gamma-secretase cleavage is performed by a transmembrane protein complex containing presenilin (PS), nicastrin (Nct), anterior pharynx defective-1 (Aph-1), presenilin enhancer-2 (Pen-2), and possibly other components. The biological understanding of gamma-secretase remains elusive, as does the mechanism by which Abeta causes neurodegeneration in AD. The work presented in this thesis has focused on studies of gamma-secretase activity and localization in mammalian brain, as well as on identification and quantification of Abeta species that are deposited in AD brains. In paper I, a detailed analysis was performed to see how active gamma-secretase is best prepared from brain material, and in what subcellular fraction the activity is highest. The gamma-secretase activity was highly affected by detergents; and the fraction containing endosomes, endoplasmic reticulum, Golgi and synaptic vesicles revealed the highest activity. It was possible to measure Abeta production under the optimized conditions. In paper II, active gamma-secretase was further studied in detergent resistant membranes (DRMs). Active gamma-secretase was localized to DRMs in human and rat brain. The size of DRMs containing active gamma-secretase, and possibly other proteins and lipids, was estimated to be > 2000 kDa. Furthermore, it was possible to measure Abeta production in DRMs. In paper III and paper IV the product of amyloidogenic gamma-secretase cleavage was studied and a detailed investigation performed on Abeta species deposited in AD brains. A method was established for quantification of C-terminal Abeta species in purified plaque cores and in total amyloid preparations fromsporadic and familial AD brains. It was found that a longer Abeta species, i.e. Abeta43, was more frequent than Abeta40. Immunohistochemistry that was performed supported these findings. In paper IV, Abeta species were quantified in six different brain regions obtained from two mutation carriers having the I143T PSEN1 mutation, reported here in Sweden for the first time. As in paper III, Abeta43 was much more frequent than Abeta40. In conclusion, we have determined the optimal conditions for studies of active gamma- secretase in brain and have showed that this active enzyme complex is localized to lipid rafts in human and rat brain. Further, we have found a longer Abeta species, Abeta43, to be more frequent than Abeta40 in amyloid depositions in AD brains. This species polymerizes rapidly, and we suggest that Abeta43 may be of importance in AD etiology.