Faculty Bibliography

Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein (Abeta) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Abeta following a reaction catalyzed by an enzyme termed 'gamma-secretase'. The molecular mechanism of PS1-mediated, gamma-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the gamma-secretase protease or whether instead PS1 mediates gamma-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DeltaE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Abeta-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DeltaE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DeltaE9 molecules expressed in Sf9 cells retain the ability to modulate Abeta levels. Baculoviral-expressed PS1DeltaE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting gamma-secretase activity from its individual purified component parts

Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by single-cell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively

Increasing evidence points to synaptic plasticity impairment as one of the first events in Alzheimer's disease (AD). However, studies on synaptic dysfunction in different transgenic AD models that overexpress familial AD mutant forms of amyloid precursor protein (APP) and/or presenilin (PS) have provided conflicting results. Both long-term potentiation (LTP) and basal synaptic transmission (BST) have been found to be both unchanged and altered in different models and under differing experimental conditions. Because of their more robust amyloid-beta (Abeta) deposition, double transgenic mice currently are used by several laboratories as an AD model. Here, we report that mice overexpressing APP (K670N:M671L) together with PS1 (M146L) have abnormal LTP as early as 3 months of age. Interestingly, reduced LTP paralleled plaque appearance and increased Abeta levels and abnormal short-term memory (working memory). BST and long-term memory (reference memory) are impaired only later (approximately 6 months) as amyloid burden increases. Abeta pathology across different ages did not correlate with synaptic and cognitive deficits, suggesting that Abeta levels are not a marker of memory decline. In contrast, progression of LTP impairment correlated with the deterioration of working memory, suggesting that percentage of potentiation might be an indicator of the cognitive decline and disease progression in the APP/PS1 mice

In Alzheimer disease (AD) patients, early memory dysfunction is associated with glucose hypometabolism and neuronal loss in the hippocampus. Double transgenic (Tg) mice co-expressing the M146L presenilin 1 (PS1) and K670N/M671L, the double 'Swedish' amyloid precursor protein (APP) mutations, are a model of AD amyloid-beta deposition (Abeta) that exhibits earlier and more profound impairments of working memory and learning than single APP mutant mice. In this study we compared performance on spatial memory tests, regional glucose metabolism, Abeta deposition, and neuronal loss in APP/PS1, PS1, and non-Tg (nTg) mice. At the age of 2 months no significant morphological and metabolic differences were detected between 3 studied genotypes. By 8 months, however, APP/PS1 mice developed selective impairment of spatial memory, which was significantly worse at 22 months and was accompanied by reduced glucose utilization in the hippocampus and a 35.8% dropout of neurons in the CA1 region. PS1 mice exhibited a similar degree of neuronal loss in CA1 but minimal memory deficit and no impairment of glucose utilization compared to nTg mice. Deficits in 22 month APP/PS1 mice were accompanied by a substantially elevated Abeta load, which rose from 2.5% +/- 0.4% at 8 months to 17.4% +/- 4.6%. These findings implicate Abeta or APP in the behavioral and metabolic impairments in APP/PS1 mice and the failure to compensate functionally for PS1-related hippocampal cell loss