Kieran C. Breen

The amyloidogenic potential and behavioral correlates of stress

Observations of elevated basal cortisol levels in Alzheimers disease (AD) patients prompted the hypothesis that stress and glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Consistent with that hypothesis, we show that stress and GC provoke misprocessing of amyloid precursor peptide in the rat hippocampus and prefrontal cortex, resulting in increased levels of the peptide C-terminal fragment 99 (C99), whose further proteolytic cleavage results in the generation of amyloid-β (Aβ). We also show that exogenous Aβ can reproduce the effects of stress and GC on C99 production and that a history of stress strikingly potentiates the C99-inducing effects of Aβ and GC. Previous work has indicated a role for Aβ in disruption of synaptic function and cognitive behaviors, and AD patients reportedly show signs of heightened anxiety. Here, behavioral analysis revealed that like stress and GC, Aβ administration causes spatial memory deficits that are exacerbated by stress and GC; additionally, Aβ, stress and GC induced a state of hyperanxiety. Given that the intrinsic properties of C99 and Aβ include neuroendangerment and behavioral impairment, our findings suggest a causal role for stress and GC in the etiopathogenesis of AD, and demonstrate that stressful life events and GC therapy can have a cumulative impact on the course of AD development and progression.

Glucocorticoids trigger Alzheimer disease-like pathobiochemistry in rat neuronal cells expressing human tau.

Amyloid precursor protein (APP) mis‐processing and aberrant tau hyperphosphorylation are causally related to the pathogenesis and neurodegenerative processes that characterize Alzheimer’s disease (AD). Abnormal APP metabolism leads to the generation of neurotoxic amyloid beta (Aβ), whereas tau hyperphosphorylation culminates in cytoskeletal disturbances, neuronal dysfunction and death. Many AD patients hypersecrete glucocorticoids (GC) while neuronal structure, function and survival are adversely influenced by elevated GC levels. We report here that a rat neuronal cell line (PC12) engineered to express the human ortholog of the tau protein (PC12‐htau) becomes more vulnerable to the toxic effects of either Aβ or GC treatment. Importantly, APP metabolism in GC‐treated PC12‐htau cells is selectively shifted towards increased production of the pro‐amyloidogenic peptide C99. Further, GC treatment results in hyperphosphorylation of human tau at AD‐relevant sites, through the cyclin‐dependent kinase 5 (E.C. 2.7.11.26) and GSK3 (E.C. 2.7.11.22) protein kinases. Pulse‐chase experiments revealed that GC treatment increased the stability of tau protein rather than its de novo synthesis. GC treatment also induced accumulation of transiently expressed EGFP‐tau in the neuronal perikarya. Together with previous evidence showing that Aβ can activate cyclin‐dependent kinase 5 and GSK3, these results uncover a potential mechanism through which GC may contribute to AD neuropathology.

Factors influencing the processing and function of the amyloid β precursor protein—a potential therapeutic target in Alzheimer's disease?

The amyloid beta precursor protein (AbetaPP), which plays a pivotal role in Alzheimers disease (AD), can exist as either a membrane-bound or soluble protein. The former is cleaved at the level of the plasma membrane to generate the soluble form of the protein (AbetaPP(s)). An alternative pathway exists, however, for the cleavage of AbetaPP to generate a 40-42 amino acid peptide termed amyloid beta (Abeta), either within the lysosomal or the endoplasmic reticulum/Golgi compartments of the cell. In AD, there is an increase in the ratio of the 42 amino acid form of the Abeta peptide (Abeta(42)) to Abeta(40). The Abeta(42) form is the more amyloidogenic form and has an increased potential to form the insoluble amyloid deposits characteristic of AD pathology. Studies on the familial form of the disease, with mutations in AbetaPP or in the presenilin proteins, have confirmed an increase in Abeta(42) generation associated with the early stages of the disease. This review will examine the factors that influence AbetaPP processing, how they may act to modulate the biological effects of AbetaPP(s) and Abeta, and if they provide a viable target for therapeutic intervention to modify the rate of progression of the disease.

The potential role of tau protein O-glycosylation in Alzheimer's disease

Single O-linked N-acetylglucosamine (O-GlcNAc) sugar residues can compete with phosphate groups to occupy specific sites on certain nuclear and cytosolic proteins. Here we show that inhibiting cellular kinase activities resulted in changes in protein O-glycosylation levels in heat-stable cytoskeletal protein fractions derived from primary neuronal cells. As increased phosphorylation of the microtubule-associated protein tau is one of the pathological hallmarks of Alzheimers disease and glycosylation may play an influential role in this process. We observed a significant decrease in the protein O-GlcNAc glycosylation of a tau-enriched cytoskeletal fraction generated from AD post-mortem brain samples as compared with control, suggesting an inverse relationship between the two post-translational modifications. Finally, cells transfected with the cDNA coding for O-GlcNAc transferase (OGT) displayed altered tau phosphorylation patterns as compared with control cells, suggesting that changes in tau glycosylation may influence its phosphorylation state. The specificity of the changes in the phosphorylation of individual amino acid residues provides evidence for a targeted O-glycosylation of tau.

The role of glycoproteins in neural development, function, and disease

Glycoproteins play key roles in the development, structuring, and subsequent functioning of the nervous system. However, the complex glycosylation process is a critical component in the biosynthesis of CNS glycoproteins that may be susceptible to the actions of toxicological agents or may be altered by genetic defects. This review will provide an outline of the complexity of this glycosylation process and of some of the key neural glycoproteins that play particular roles in neural development and in synaptic plasticity, in the mature CNS. Finally, the potential of glycoproteins as targets for CNS disorders will be discussed.

Synaptosomal sialyltransferase glycosylates surface proteins that are inaccessible to the action of membrane-bound sialidase.

Abstract Sialyltransferase has been characterized in P2 pellets derived from animals of increasing age. The enzyme was found to be associated with the plasma membrane and to be developmentally regulated at times coincident with cell migration and fibre outgrowth. This regulation appeared to be due, in part, to an endogenous competitive inhibitor in the P2 pellet but not in the synaptosome. Optimal transfer of [14C]N‐acetylneuraminic acid to endogenous synaptosomal acceptors was achieved only in the absence of detergent. Furthermore, the transferred sialic acid was found to be inaccessible to the action of membrane‐bound sialidase. The significance of these findings is discussed.

The dynamic localization of the glucocorticoid receptor in rat C6 glioma cell mitochondria

Glucocorticoids modify gene expression via the translocation of receptors from the cytosol to the nucleus following agonist-associated receptor activation. In this study, we have characterized mitochondrial glucocorticoid (GR) localization and associated translocation kinetics in the C6 mouse glioma cell line. Treatment of the cells, which were cultured in steroid-depleted culture medium, with the GR agonist dexamethasone (dex) resulted in a dramatic decrease in mitochondrial GR levels in parallel with those of the cytosolic receptor. The effect was not observed in isolated intact mitochondria suggesting that the effect is unlikely to be direct but is rather a component of the combined cellular response to GR activation. A marked stimulation of the expression of the mitochondrially-encoded cytochrome oxidase-1 (COX-1) gene was found following GR activation and its export from mitochondria. The effects were inhibited by RU486. Therefore, GR is likely to have a functional role at the level of the mitochondria within intact cells.

Postnatal D2‐CAM/N‐CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi‐enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2‐CAM/N‐CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12‐day‐old animals the majority of the transferred [14C]sialic acid was found to be associated with the high‐molecular‐weight [>200 kilodaltons (kd)] form of D2‐CAM/N‐CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2‐CAM/N‐CAM polypeptides was also demonstrated in both 12‐day and adult animals and transfer was evident only in the 180‐kd and 115‐kd components and not in the 140‐kd component. In contrast, Golgi‐enriched fractions prepared from adult animals showed little capability of heavily sialylating D2‐CAM/N‐CAM to any significant extent.

Autoinsertion of soluble oligomers of Alzheimer's Aβ(1–42) peptide into cholesterol-containing membranes is accompanied by relocation of the sterol towards the bilayer surface

BackgroundSoluble Alzheimers Aβ oligomers autoinsert into neuronal cell membranes, contributing to the pathology of Alzheimers Disease (AD), and elevated serum cholesterol is a risk factor for AD, but the reason is unknown. We investigated potential connections between these two observations at the membrane level by testing the hypothesis that Aβ(1–42) relocates membrane cholesterol.ResultsOligomers of Aβ(1–42), but not the monomeric peptide, inserted into cholesterol-containing phosphatidylcholine monolayers with an anomalously low molecular insertion area, suggesting concurrent lipid rearrangement. Membrane neutron diffraction, including isomorphous replacement of specific lipid hydrogens with highly-scattering deuterium, showed that Aβ(1–42) insertion was accompanied by outward displacement of membrane cholesterol, towards the polar surfaces of the bilayer. Changes in the generalised polarisation of laurdan confirmed that the structural changes were associated with a functional alteration in membrane lipid order.ConclusionCholesterol is known to regulate membrane lipid order, and this can affect a wide range of membrane mechanisms, including intercellular signalling. Previously unrecognised Aβ-dependent rearrangement of the membrane sterol could have an important role in AD.

The role of the protein glycosylation state in the control of cellular transport of the amyloid β precursor protein

The amyloid beta precursor protein can exist as both a membrane-bound and a secreted protein, with the former having the potential to generate the amyloid beta peptide present in the neuritic plaques which are characteristic of Alzheimers disease. In this study, we have used a clone of the AtT20 mouse pituitary cell line which expresses high levels of the amyloid beta precursor protein to characterize the glycosylation state of the secreted and membrane-bound forms of the protein and to examine the role of post-translational modifications in protein processing. Lectin blot analysis of immunoprecipitated amyloid beta precursor protein demonstrated that the soluble form of the protein contains significant amounts of sialic acid, with the lectin staining being reduced in the particulate cellular fractions. Treatment of the cells with mannosidase inhibitors to interfere with the formation of complex-type N-linked glycans resulted in a decrease in secreted amyloid beta precursor protein and an increase in the level of the cellular form of the protein. The increase in amyloid beta precursor protein levels in the cellular fraction was accompanied by an increase in perinuclear staining. Furthermore, cells overexpressing the alpha2,6(N)-sialyltransferase enzyme also demonstrated an increase in amyloid beta precursor protein secretion. These results suggest that the presence of terminal sialic acid residues on complex-type N-glycans may be required for the optimal transport of the amyloid beta precursor protein from the Golgi to the cell membrane with the subsequent cleavage to generate the secreted form of the protein.