The amyloidogenic potential and behavioral correlates of stress.

Observations of elevated basal cortisol levels in Alzheimer's 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-beta (Abeta). We also show that exogenous Abeta can reproduce the effects of stress and GC on C99 production and that a history of stress strikingly potentiates the C99-inducing effects of Abeta and GC. Previous work has indicated a role for Abeta 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, Abeta administration causes spatial memory deficits that are exacerbated by stress and GC; additionally, Abeta, stress and GC induced a state of hyperanxiety. Given that the intrinsic properties of C99 and Abeta 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.

The over-expression of the wild type or mutant forms of the presenilin-1 protein alters glycoprotein processing in a human neuroblastoma cell line.

Mutations in the presenilin proteins (PS1 and PS2) are responsible for more than 70% of the cases of the familial form of Alzheimer's disease (FAD). The proteins are expressed in the cell at a low level, primarily in the endoplasmic reticulum and cis Golgi, where they have been proposed to play a role in protein processing. As protein glycosylation is a key post-translational event that occurs within the Golgi, we have investigated the effect of altered PS1 expression levels on the protein glycosylation pattern using the SH-SY5Y human neuroblastoma cell line. In cells over-expressing either the wild type or mutant (M146L) PS1-FAD proteins, there was a decrease in the expression levels of protein-bound alpha2,3-linked sialic acid residues at the level of the cell membrane. This was particularly manifest as a significant decrease in the expression of the polysialic acid chain that is linked to the core oligosaccharide of the neural cell adhesion molecule in an alpha2,3 bond. These results suggest that the over-expression of either the wild type or mutant PS1 disturbs glycoprotein processing within the Golgi.

The role of post-translational modification in beta-amyloid precursor protein processing.

The beta-amyloid precursor protein (APP) plays a pivotal role in the early stages of neurodegeneration associated with Alzheimer's disease. An alteration in the processing pattern of the protein results in an increase in the generation of the 40-42-amino-acid beta-amyloid (A beta) peptide, which coalesces to form insoluble, extracellular amyloid deposits. A greater understanding of the factors that influence APP processing may assist in the design of effective therapeutic agents to halt progression of Alzheimer's disease. APP is a sialoglycoprotein with two potential N-linked glycosylation sites, one of which may contain a complex oligosaccharide chain. An alteration in the glycosylation state of APP by the generation of oligomannosyl oligosaccharides results in a decrease in the secretion of the neuroprotective, soluble form of the protein and a parallel increase in the deposition of the cellular protein within the perinuclear region of the cell. Conversely, the attachment of additional terminal sialic acid residues on to the oligosaccharide chain results in an increase in secretion of soluble APP (sAPP alpha). One factor that has been widely reported to alter APP processing is the activation of protein kinase C (PKC). This process has been characterized using synaptosomal preparations, which suggests that the PKC action is occurring at the level of the plasma membrane. Furthermore, when cells are transfected with the sialyltransferase enzyme, there is a direct relationship between the sialylation potential of APP and the fold stimulation of sAPP alpha, after PKC activation. These results suggest that the post-translational modification of APP by glycosylation is a key event in determining the processing of the protein.

Activation of phospholipase D by metabotropic glutamate receptor agonists in rat cerebrocortical synaptosomes.

The pharmacological profile of metabotropic glutamate receptor (mGluR) activation of phospholipase D (PLD), and the associated signalling pathways, were examined in rat cerebrocortical synaptosomes. The assay was conducted using a transphosphatidylation reaction in synaptosomes which were pre-labelled with either [(3)H]-arachidonic acid or [(32)P]-orthophosphate. The mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD) and (RS)-3,5-dihydroxyphenylglycine (DHPG), both activated PLD, while phorbol 12,13-dibutyrate (PDBu) treatment caused receptor-independent activation of PLD and had an additive effect on 1S,3R-ACPD induced PLD activity. A protein kinase C (PKC) inhibitor, GF109203X, failed to antagonize mGluR receptor-coupled PLD activity. We could not detect any increase in the products of PI (phosphoinositide)-specific phospholipase C (PI-PLC), inositol(1,4, 5)trisphosphate or diacylglycerol, by 1S, 3R-ACPD at 15 s. However, diacylglycerol increased monophasically in response to mGluR agonists and remained elevated for at least 15 min. Phosphatidic acid phosphohydrolase (PAP) activity, which converts PA to DAG, was present in the synaptosomes. These data suggest that, in rat cerebrocortical synaptosomes, the 1S,3R-ACPD-sensitive mGluR is coupled to PLD through a mechanism that is independent of both PKC and PI-PLC.

Inhibition of N-glycan processing alters axonal transport of synaptic glycoproteins in vivo.

Synaptic glycoproteins are synthesized and glycosylated in the neuronal cell body, and conveyed to terminals by fast axonal transport. We used the alpha-mannosidase inhibitor, 2-deoxymannojirimycin (dMan), to investigate the effects of disrupting N-glycan processing on the axonal trafficking of proteins in vivo. dMan significantly reduced rapid axonal transport in retinal ganglion cells to about 34% of control values 4h after metabolic labeling; at 8 h post-labeling the inhibition was reversed. 2-D gel analysis showed that dMan completely inhibited the arrival of radiolabeled L1 and NCAM at axon terminals, and resulted in the appearance of two novel proteins of 230 kDa and 155 kDa. Our results show that disruption of the N-glycosylation pathway has an immediate inhibitory effect on total axonal transport and longer lasting effects on the trafficking of specific glycoproteins to axon terminals in vivo.

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

The amyloid beta precursor protein (AbetaPP), which plays a pivotal role in Alzheimer's 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.

Retinoic acid induction of sialyltransferase activity in neuroblastoma cells of differing sialylation potentials.

In order to determine how glycosylation changes associated with cellular differentiation may be influenced by the basal cellular sialylation potential, the effect of retinoic acid (RA)-induced differentiation was investigated in neuroblastoma cells expressing differing levels (and activities) of the alpha2,6(N) sialyltransferase (ST6N) enzyme. The increase in ST activity was proportional to the basal cellular sialylation potentials with the high activity clones showing the greatest increase. This was paralleled by an up-regulation of the level of overall sialoglycoprotein glycosylation level. An increase in the levels of the polysialic acid (PSA) epitope was associated with a parallel increase in the levels of the neural cell adhesion molecule (NCAM) protein backbone although there was no overall change in the PSA:NCAM ratio following RA treatment.

The effect of corticosteroids on amyloid beta precursor protein/amyloid precursor-like protein expression and processing in vivo.

In this study, we have investigated the effect of altered corticosteroid levels on the expression and processing of the amyloid beta precursor protein (A betaPP) and its amyloid precursor-like protein (APLP) homologue in rat brain. Four groups of animals were used in the study: sham operated, adrenalectomised, and adrenalectomised treated with either dexamethasone or aldosterone, with the A betaPP/APLP expression being determined by western blot analysis. While there were no changes in the levels of A betaPP/APLP following adrenalectomy, treatment with dexamethasone, but not aldosterone, resulted in a marked increase in protein expression levels with the level of increase varying between the brain regions examined. Corticosteroids had a more marked effect on the particulate rather than the soluble form of the protein, thus suggesting that elevated glucocorticoids may also be adversely influencing A betaPP/APLP processing.

Overexpression of the alpha2,6 (N) sialyltransferase enzyme in human and rat neural cell lines is associated with increased expression of the polysialic acid epitope.

The function of the neural cell adhesion molecule, NCAM, is modulated by the expression of the N-linked polysialic acid (PSA) oligosaccharide chain, with PSA serving to decrease the adhesive potential of the protein backbone. In this study, we have generated clonal cells of the rat B104 and human SH-SY5Y neuroblastoma cell lines that over-express the alpha2,6(N) sialyltransferase (ST6N) enzyme in order to investigate the role of this enzyme in PSA biosynthesis. The clonal cells exhibited ST enzyme activities of up to 20-times control levels, which remained stable throughout the duration of the study. The increase in enzyme activity paralleled an increase in enzyme protein levels, as determined by Western blot analysis, and immunocytochemical analysis confirmed the Golgi localisation of the enzyme. The induction of PSA-NCAM expression in the cells expressing high levels of ST6N was confirmed both by using anti-PSA antisera and by specific digestion with endo-N-acetylneuraminidase E, whose actions are specific for alpha2, 8-linked PSA chains. These results demonstrate that the cellular ST6N activity serves to positively influence the expression of PSA in neuronal cells.

Modest cholinergic deafferentation fails to alter hippocampal G-proteins.

The integrity of hippocampal G-protein mediated signalling following ibotenate induced lesion of the medial septum was examined. The lesion was confined histologically to the septum and induced a 23% reduction in hippocampal choline acetyltransferase (ChAT) activity and G-proteins levels and related enzyme activities were measured in the hippocampus following a 21 day survival period. The relative levels of five G-protein subunits (Gbeta, G(alpha)o, G(alpha)i1, G(alpha)i2, and G(alpha)s-L), basal GTPase, the degree of carbachol- or baclofen-stimulated GTPase activities, and the basal and fluoroaluminate-stimulated adenylate cyclase activities were apparently unaffected. To determine if our assay methodology was sensitive to changes in pre-synaptic signalling, we compared G-protein density in synaptosomes with total hippocampal homogenates. The concentration of G(alpha)q/11, G(alpha)i1, and G(alpha)i2. were significantly lower in synaptosomes, while G(alpha)o, was only marginally reduced. Thus, modest lesions of the medial-septal nucleus fail to alter G-protein signalling. However, our findings that G-protein density is lower in synaptosomal membranes than in total homogenates, indicates that the analysis of signalling events in synaptosomes following deafferentation could clarify adaptive changes which may occur at the presynaptic level.

The role of the protein glycosylation state in the control of cellular transport of the amyloid beta 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 Alzheimer's 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.

Protein kinase C activation potentiates the rapid secretion of the amyloid precursor protein from rat cortical synaptosomes.

In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AbetaPP) by the alpha-secretase pathway within the betaA4 domain to generate a soluble secreted N-terminal fragment (AbetaPPs). AbetaPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform beta1 and novel PKCepsilon from cytosol to membrane fractions, but there was no alteration in the proportion of AbetaPP associated with the Triton-soluble and -insoluble fractions. AbetaPPs was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AbetaPPs was only partially blocked by the PKC inhibitor GF109203X (2.5 microM), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AbetaPPs secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the alpha-secretase activity leading to the secretion of AbetaPPs can occur at the level of the presynaptic terminal.

Overexpression of alpha2,3 sialyltransferase in neuroblastoma cells results in an upset in the glycosylation process.

Glycosylation is key posttranslational modification for membrane-bound and secreted proteins that can influence both the secondary structure and the function of the protein backbone. In order to investigate the effect of altered cellular glycosylation potential, we have generated a number of clonal cell lines over-expressing the alpha2,3(N) sialyltransferase enzyme (ST3N). In general, there was a decrease in total sialyltransferase (ST) enzyme activity in the clones transfected with the ST3N cDNA, with this decrease being inversely proportional to the quantity of the mRNA coding for the enzyme. The ST3N enzyme was, however, functional and there was an increase in both MAA lectin staining and the expression of polysialic acid, which is attached to the NCAM protein backbone primarily via an alpha2,3 linkage. These results suggest that the overexpression of a sialyltransferase may upset the sialylation potential of the cell.

Stimulation of sialyltransferase by subchronic low-level lead exposure in the developing nervous system. A potential mechanism of teratogen action.

Chronic low-level lead exposure has been associated with mental deficits in young children, possibly due to its actions on specific targets in the developing nervous system. Protein glycosylation has been demonstrated to play a critical role during CNS development, and the negatively charged sialic acid group has been particularly associated with the modulation of cell adhesion. In this study, we have used an in vitro model system to examine the effect of subchronic low-level lead on the expression and activity of the sialyltransferase (ST) enzyme family. Subchronic exposure of neuronal cells to low concentrations of lead (10(-6)-10(-16) M) resulted in up to a 3-fold induction of total cellular ST activity, the level of induction being more pronounced in embryonically derived cells compared with postnatally derived cells. The increase was not due to a direct interaction of the metal with the enzyme and was only observed after at least 72 h exposure to the metal. The induction was blocked by the protein synthesis inhibitor, cycloheximide, and could be reversed upon removal of the metal. The increase was due primarily to the induction of the alpha2,3(N) ST enzyme with no effect on the expression of the alpha2,6(N) enzyme. These results suggest that the ST enzyme may serve as a target for the actions of chronic low-level lead in vivo with an alteration in the developmental regulation of protein glycosylation being at least partially responsible for the behavioral deficits associated with toxin exposure.

The interaction between chronic low-level lead and the amyloid beta precursor protein.

Chronic low-level lead exposure is toxic to the developing nervous system. The amyloid beta precursor protein (A beta PP) plays a pivotal role in this developmental process, both as a neurotrophic/neuroprotective factor and as a mediator of cell adhesion. In this study, we have used an in vitro system to examine the interaction between chronic low-level lead and the expression and function of A beta PP. Chronic exposure of the HN9 mouse hippocampal cell line to lead chloride (10(-14) M to 10(-6) M) for 96 hours resulted in a 50% increase in the levels of the particulate form of the protein with a parallel decrease in the soluble form (A beta PP). This effect of lead was reversible following the removal of the toxin. This increase in membrane-bound A beta PP was also paralleled by an increase in cell adhesivity to a fibronectin substrate. In addition, A beta PP also acted to attenuate lead toxicity. Cells which secreted high levels of the protein were resistant to lead toxicity when compared with control cells suggesting that the protein may be acting to chelate the metal and thus attenuating its toxic action within the cell.

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.

The generation and characterization of a rat neural cell line overexpressing the alpha2,6(N) sialyltransferase.

In order to examine the effects of altered protein sialylation on neural cell function, B104 rat neuroblastoma cells were stably transfected with the cDNA coding for alpha2,6(N) sialyltransferase (ST(6)N). Lectin blot analysis of the clones demonstrated an increase in staining of the Sambucus nigra lectin, which detects alpha2,6 linked sialic acid, in parallel with enzyme activity. There was a concomitant decrease in staining by the Maackia amurensis lectin which labels alpha2,3-linked sialic acid, indicating that the individual sialyltransferase enzymes may compete for penultimate galactose acceptor sites. While there was an initial increase in protein-bound sialic acid in parallel with enzyme activity, the sialylation of the cells was demonstrated to be saturable. There was an inverse relationship between cell adhesion to a fibronectin substrate and ST(6)N activity suggesting that the negatively charged sugar acts to modulate cell-substrate interaction. These cells will provide an ideal model system with which to further investigate the effect of altered sialic acid on neural cell function.

Glucocorticoid induction of the alpha2,6 sialyltransferase enzyme in a mouse neural cell line.

Combined sialyltransferase (ST) activities were induced in the HN9 hippocampal cell line following treatment with the synthetic glucocorticoid dexamethasone (dex) for 24 hr. Induction occurred in a dose-dependent manner, with the maximum induction of a 2-fold increase over control enzyme levels occurring at a concentration of 10(-8) M dex. A minimum of 6 hr pretreatment with drug was required before significant induction could be detected and elevated enzyme levels persisted for up to 48 hr post-treatment. The induced form of the enzyme showed an increase in reaction maximum velocity (Vmax) while showing no change in affinity (Km) for the acceptor substrate asialofetuin. The alpha2,6 ST enzyme was demonstrated to be the primary enzyme induced and there was no change in expression of the alpha2,3 ST enzyme. Lectin blot analysis demonstrated an increase in the levels of the alpha2,6-linked cellular sialoglycoproteins and a parallel decrease in the alpha2,3 sialoglycoprotein levels.

The effect of corticosteroids on serum sialyltransferase enzyme activities in the rat.

Previous studies have demonstrated corticosteroid regulation of sialyltransferase (sialyl-T) enzyme activities in a number of different tissues throughout the body. In this study we examined the regulatory effect of corticosteroids on serum enzyme activity in the rat. The total serum sialyl-T activity was not affected by a decrease in corticosteroid levels following adrenalectomy. However, while there was a significant increase in enzyme activity following dexamethasone treatment, aldosterone had no effect on this parameter. Subsequent examination of individual sialyl-T enzymes demonstrated a slight decrease in alpha2,6 sialyl-T activity following adrenalectomy which was restored to basal levels following dexamethasone treatment. The activity of the alpha2,3 sialyl-T enzyme was not affected by adrenalectomy or dexamethasone treatment, but was stimulated significantly by aldosterone. In general, the levels of serum sialoglycoproteins mirrored well the activities of the appropriate sialyl-T enzymes. These results demonstrate that serum sialyltransferase activity in the rat is under the influence of circulating corticosteroids.

The biochemical consequences of alpha2,6(N) sialyltransferase induction by dexamethasone on sialoglycoprotein expression in the rat H411e hepatoma cell line.

Previous studies have demonstrated sialyltransferase (ST) enzyme activity to be induced in hepatic cells by corticosteroids. In this study, we used the H411e rat hepatoma cell line to further characterise this induction with particular reference to the subsequent changes in the pattern of sialoglycoprotein (SGP) expression. The induction of total ST activity by dexamethasone was concentration dependent with maximum induction occurring 12 h subsequent to drug addition. Western blot analysis demonstrated that the induction was associated with an increase in the expression of the alpha2,6(N) ST enzyme with no change in the expression levels of the alpha2,3(N) enzyme. While the induction resulted in an increase in the reaction velocity (Vmax) of the enzyme for both the sugar donor (CMP-Neu5Ac) and the asialofetuin acceptor protein, there was no significant change in the enzyme affinity (Km) for the substrates, suggestive of either an increase in the expression or efficiency of the existing enzyme(s) rather than an induction of novel ST enzymes. Lectin blot analysis of cellular glycoprotein expression demonstrated no change in the expression patterns of either alpha2,3 or alpha2,6-linked SGP following enzyme induction. These results suggest that the available acceptor sites for the terminal sialic acid group(s) may be fully occupied in the control cells and therefore there are no further sites onto which the sialic acid can be transferred following induction of ST enzyme activity. This may be due to the high basal enzyme levels in the control cells already exhausting endogenous acceptor sites.