Barbara Cordell

Phosphorylation, Subcellular Localization, and Membrane Orientation of the Alzheimer's Disease-associated Presenilins

Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37°C instead of 95°C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 → Glu or Cys-410 → Tyr that cause Alzheimers disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.

Inhibition of β-amyloid formation identifies proteolytic precursors and subcellular site of catabolism

Cerebral deposition of beta-amyloid protein is a pathological feature central to Alzheimers disease. Production of beta-amyloid by proteolytic processing of the beta-amyloid precursor protein (beta APP) is a critical initial step in beta-amyloidogenesis. We use an inhibitor of beta APP processing to block beta-amyloid peptide formation. Application of the inhibitor to cultured cells results in an accumulation of proteolytic intermediates of beta APP, enabling a precursor-product relationship between beta APP carboxy-terminal fragments and beta-amyloid peptides to be demonstrated directly. In the presence of inhibitor, these amyloidogenic carboxy-terminal fragments can be degraded to nonamyloidogenic products. The catabolism of beta APP carboxy-terminal intermediates and the formation of beta-amyloid peptides are likely to involve an early endosomal compartment as the subcellular site of processing.

Specific spatial learning deficits become severe with age in β-amyloid precursor protein transgenic mice that harbor diffuse β-amyloid deposits but do not form plaques

Memory impairment progressing to dementia is the main clinical symptom of Alzheimers disease (AD). AD is characterized histologically by the presence of β-amyloid (Aβ) plaques and neurofibrillary tangles in specific brain regions. Although Aβ derived from the Aβ precursor protein (β-APP) is believed to play a central etiological role in AD, it is not clear whether soluble and/or fibrillar forms are responsible for the memory deficit. We have generated and previously described mice expressing human wild-type β-APP751 isoform in neurons. These transgenic mice recapitulate early histopathological features of AD and form Aβ deposits but no plaques. Here we describe a specific and progressive learning and memory impairment in these animals. In the Morris water maze, a spatial memory task sensitive to hippocampal damage, one pedigree already showed significant differences in acquisition in 3-month-old mice that increased in severity with age and were expressed clearly in 6-month- and 2-year-old animals. The second transgenic pedigree displayed a milder impairment with a later age of onset. Performance deficits significantly decreased during the 6 days of training in young but not in aged transgenic animals. Both pedigrees of the transgenic mice differed from wild-type mice by less expressed increase of escape latencies after the platform position had been changed in the reversal experiment and by failure to prefer the goal quadrant in probe trials. Both pedigrees performed at wild-type level in a number of other tests (open field exploration and passive and active place avoidance). The results suggest that plaque formation is not a necessary condition for the neuronal β-APP751 transgene-induced memory impairment, which may be caused by β-APP overexpression, isoform misexpression, or elevated soluble Aβ.

Positive and negative regulation of APP amyloidogenesis by sumoylation

Amyloid β peptide (Aβ) generated from amyloid precursor protein (APP) is central to Alzheimers disease (AD). Signaling pathways affecting APP amyloidogenesis play critical roles in AD pathogenesis and can be exploited for therapeutic intervention. Here, we show that sumoylation, covalent modification of cellular proteins by small ubiquitin-like modifier (SUMO) proteins, regulates Aβ generation. Increased protein sumoylation resulting from overexpression of SUMO-3 dramatically reduces Aβ production. Conversely, reducing endogenous protein sumoylation with dominant-negative SUMO-3 mutants significantly increases Aβ production. We also show that mutant SUMO-3, K11R, which can only be monomerically conjugated to target proteins, has an opposite effect on Aβ generation to that by SUMO-3, which can form polymeric chains on target proteins. In addition, SUMO-3 immunoreactivity is predominantly detected in neurons in brains from AD, Downs syndrome, and nondemented humans. Therefore, polysumoylation reduces whereas monosumoylation or undersumoylation enhances Aβ generation. These findings provide a regulatory mechanism in APP amyloidogenesis and suggest that components in the sumoylation pathway may be critical in AD onset or progression.

Macrophage colony-stimulating factor augments beta-amyloid-induced interleukin-1, interleukin-6, and nitric oxide production by microglial cells.

In Alzheimer’s disease (AD), a chronic cerebral inflammatory state is thought to lead to neuronal injury. Microglia, intrinsic cerebral immune effector cells, are likely to be key in the pathophysiology of this inflammatory state. We showed that macrophage colony-stimulating factor, a microglial activator found at increased levels in the central nervous system in AD, dramatically augments β-amyloid peptide (βAP)-induced microglial production of interleukin-1, interleukin-6, and nitric oxide. In contrast, granulocyte macrophage colony-stimulating factor, another hematopoietic cytokine found in the AD brain, did not augment βAP-induced microglial secretory activity. These results indicate that increased macrophage colony-stimulating factor levels in AD could magnify βAP-induced microglial inflammatory cytokine and nitric oxide production, which in turn could intensify the cerebral inflammatory state by activating astrocytes and additional microglia, as well as directly injuring neurons.

Neuroinflammation and Alzheimer’s disease: critical roles for cytokine/Aβ-induced glial activation, NF-κB, and apolipoprotein E

Neuroscience Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA Departments of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, MO 63110, USA Scios, Inc., Sunnyvale, CA 94086, USA

Behavioral Disturbances without Amyloid Deposits in Mice Overexpressing Human Amyloid Precursor Protein with Flemish (A692G) or Dutch (E693Q) Mutation

The contribution of mutations in the amyloid precursor protein (APP) gene known as Flemish (APP/A692G) and Dutch (APP/E693Q) to the pathogenesis of Alzheimers disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type, respectively, was studied in transgenic mice that overexpress the mutant APP in brain. These transgenic mice showed the same early behavioral disturbances and defects and increased premature death as the APP/London (APP V717I), APP/Swedish (K670N, M671L), and other APP transgenic mice described previously. Pathological changes included intense glial reaction, extensive microspongiosis in the white matter, and apoptotic neurons in select areas of the brain, while amyloid deposits were absent, even in mice over 18 months of age. This contrasts with extensive amyloid deposition in APP/London transgenic mice and less pronounced amyloid deposition in APP/Swedish transgenic mice generated identically. It demonstrated, however, that the behavioral deficiencies and the pathological changes in brain resulting from an impaired neuronal function are caused directly by APP or its proteolytic derivative(s). These accelerate or impinge on the normal process of aging and amyloid deposits per se are not essential for this phenotype.

Structure and processing of yeast precursor tRNAs containing intervening sequences.

We have isolated a precursor of yeast tRNATyr and shown that it contains an intervening sequence identical to that found in the gene for tRNATyr. The conformation of pre-tRNATyr is similar to that of mature tRNATyr except for the anticodon loop. The loop is sensitive to endonucleolytic cleavage by S1 nuclease near to the ends of the intervening sequence. This pre-tRNA is functionally inactive as it cannot be aminoacylated and the anticodon is not accessible for hydrogen bonding. A crude nuclear extract from yeast contains an excision-ligase activity which will process pre-tRNATyr into mature tRNATyr.

BETA -AMYLOID PEPTIDE SECRETION BY A MICROGLIAL CELL LINE IS INDUCED BY BETA -AMYLOID-(25-35) AND LIPOPOLYSACCHARIDE

β-Amyloid protein (βAP) deposition is a neuropathologic hallmark of Alzheimers disease (AD). Yet, the source of cerebral βAP in AD is controversial. We examined the production of βAP by the BV-2 immortalized microglial cell line using a sensitive enzyme immunoassay. Constitutive production of βAP was detected in conditioned media from unstimulated BV-2 cells. Further, production of βAP was induced by treatment of cultures by lipopolysaccharide (LPS) or βAP-(25–35) and was inhibited by the calpain protease inhibitor MDL 28170. Treatment of BV-2 cells with LPS or βAP-(25–35) did not affect cell-associated β-amyloid precursor protein levels. These findings suggest that microglia may be an important source of βAP in AD, and that microglial production of βAP may be augmented by proinflammatory stimuli or by βAP itself.

Substitution of a Glycogen Synthase Kinase-3β Phosphorylation Site in Presenilin 1 Separates Presenilin Function from β-Catenin Signaling

The majority of cases with early onset familial Alzheimers disease have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 protein is a component of a high molecular weight membrane-bound complex that also contains β-catenin. The physiological relevance of the association between PS1 and β-catenin remains controversial. In this study, we report the identification and functional characterization of a highly conserved glycogen synthase kinase-3β consensus phosphorylation site within the hydrophilic loop domain of PS1. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, indicates that PS1 residues Ser353 and Ser357 are glycogen synthase kinase-3β targets. Substitution of one or both of these residues greatly reduces the ability of PS1 to associate with β-catenin. By disrupting this interaction, we demonstrate that the association between PS1 and β-catenin has no effect on Aβ peptide production, β-catenin stability, or cellular susceptibility to apoptosis. Significantly, in the absence of PS1/β-catenin association, we found no alteration in β-catenin signaling using induction of this pathway by exogenous expression of Wnt-1 or β-catenin and a Tcf/Lef transcriptional assay. These results argue against a pathologically relevant role for the association between PS1 and β-catenin in familial Alzheimers disease.