Alpha7-nicotinic acetylcholine receptors mediate an Abeta(1-42)-induced increase in the level of acetylcholinesterase in primary cortical neurones.

The beta-amyloid protein (Abeta) is the major protein component of amyloid plaques found in the Alzheimer brain. Although there is a loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurones in the brain of Alzheimer patients, the level of AChE is increased around amyloid plaques. Previous studies using P19 cells in culture and transgenic mice which overexpress human Abeta have suggested that this increase may be due to a direct action of Abeta on AChE expression in cells adjacent to amyloid plaques. The aim of the present study was to examine the mechanism by which Abeta increases levels of AChE in primary cortical neurones. Abeta1-42 was more potent than Abeta1-40 in its ability to increase AChE in primary cortical neurones. The increase in AChE was unrelated to the toxic effects of the Abeta peptides. The effect of Abeta1-42 on AChE was blocked by inhibitors of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) as well as by inhibitors of L- or N-type voltage-dependent calcium channels (VDCCs), whereas agonists of alpha7 nAChRs (choline, nicotine) increased the level of AChE. The results demonstrate that the effect of Abeta1-42 on AChE is due to an agonist effect of Abeta1-42 on the alpha7 nAChR.

Glycosylation of acetylcholinesterase and butyrylcholinesterase changes as a function of the duration of Alzheimer's disease.

The identification of biochemical markers of Alzheimer's disease (AD) may help in the diagnosis of the disease. Previous studies have shown that Abeta(1-42) is decreased, and tau and phospho-tau are increased in AD cerebrospinal fluid (CSF). Our own studies have identified glycosylated isoforms of acetylcholinesterase (Glyc-AChE) and butyrylcholinesterase (Glyc-BuChE) that are increased in AD CSF. Glyc-AChE is increased in APP (SW) Tg2576 transgenic mice prior to amyloid plaque deposition, which suggests that Glyc-AChE may be an early marker of AD. The aim of this study was to determine whether Glyc-AChE or Glyc-BuChE is increased in CSF at early stages of AD and to compare the levels of these markers with those of Abeta(1-42), tau and phospho-tau. Lumbar CSF was obtained ante mortem from 106 non-AD patients, including 15 patients with mild cognitive impairment (MCI), and 102 patients with probable AD. Glyc-AChE, tau and phospho-tau were significantly increased in the CSF of AD patients compared to non-neurological disease (NND) controls. Abeta(1-42) was lower in the AD patients than in NND controls. A positive correlation was found between the levels of Glyc-AChE or Glyc-BuChE and disease duration. However, there was no clear correlation between the levels of tau, phospho-tau or Abeta(1-42) and disease duration. The results suggest that Glyc-AChE and Glyc-BuChE are unlikely to be early markers of AD, although they may have value as markers of disease progression.

Wheat germ agglutinin-binding glycoproteins are decreased in Alzheimer's disease cerebrospinal fluid.

A number of biomarkers (e.g. Abeta, tau) has been identified in Alzheimer's disease CSF. However, none fulfils the criteria of sensitivity and specificity (> 80%) needed for the development of an accurate diagnostic test. The lack of a suitable marker has prompted the search for new CSF biomarkers. In this study, the glycosylation of CSF proteins was examined using lectin blotting. Lumbar CSF was collected ante mortem from 22 non-Alzheimer's disease and 12 probable Alzheimer's disease cases and ventricular CSF collected post mortem from 7 non-Alzheimer's disease and 16 Alzheimer's disease cases confirmed by pathologic examination. When CSF glycoproteins were stained with wheat germ agglutinin (WGA), the staining intensity was found to be significantly lower in the Alzheimer's disease group. No difference in staining was found using other lectins (Canavalia ensiformis agglutinin, Ricinus communis agglutinin, Lens culinaris agglutinin). The measurement of WGA-reactive glycoproteins in CSF may be a useful biomarker for diagnosis of Alzheimer's disease.

The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase.

The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.

Altered glycosylation of cerebrospinal fluid butyrylcholinesterase in Alzheimer's disease.

Our previous studies have shown that a minor isoform of acetylcholinesterase (AChE) is increased in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients. In the present study, the glycosylation of butyrycholinesterase (BuChE) was found to be altered in AD CSF. By combining an analysis of CSF AChE and BuChE glycosylation, it is possible to identify cases of AD with more than 90% sensitivity and specificity.

Altered glycosylation of acetylcholinesterase in lumbar cerebrospinal fluid of patients with Alzheimer's disease.

As clinical diagnosis of Alzheimer's disease is only 80%-90% accurate, there is a need to identify biochemical markers of Alzheimer's disease. Previous studies have shown an abnormality in the glycosylation of acetylcholinesterase (AChE) in the CSF collected postmortem from patients with Alzheimer's disease. This abnormality was very specific for Alzheimer's disease, as it was not detected in other illnesses causing dementia. We report here that the glycosylation of AChE is also altered in lumbar CSF collected antemortem. The altered glycosylation was due to increased concentrations of a minor AChE isoform that does not bind to concanavalin A (Con A). Glycosylation of AChE may eventually be of diagnostic value, especially when used in combination with other CSF markers.

Regulation of APP cleavage by alpha-, beta- and gamma-secretases.

Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. alpha-Secretases cleave APP within the amyloid sequences, whereas beta- and gamma-secretases cleave on the N- and C-terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as beta-secretase and several proteases (ADAM-10, TACE, PC7) may be alpha-secretases. A number of studies have suggested that presenilins could be gamma-secretases, although this remains to be demonstrated conclusively. Inhibition of beta- and gamma-secretase, or stimulation of alpha-secretase, is a rational strategy for therapeutic intervention in AD.

A beta peptides and calcium influence secretion of the amyloid protein precursor from chick sympathetic neurons in culture.

The major constituent of amyloid plaques in the Alzheimer disease (AD) brain is the amyloid protein (A beta). A beta has been shown to be neurotoxic to cells, but the exact mechanism of its effects are still not known. Most studies have focussed on A beta neurotoxicity, but little is known about the effect of A beta peptides on cellular protein metabolism and secretion. To examine the effect of A beta peptides on APP secretion, chick sympathetic neurons were metabolically labeled with [(35)S]methionine and the amounts of radiolabeled APP and A beta quantitated. Several A beta peptides (A beta(25-35), [pyroglu(3)]A beta(3-40), and [pyroglu(11)]A beta(11-40)) inhibited secretion of [(35)S]APP and increased cell-associated [(35)S]APP. There was also a 2-2.5-fold increase in secretion of several other proteins when cells were incubated with A beta(25-35). However, the amount of A beta secreted into the medium was decreased. Treatment of cells with the calcium ionophore A23187 caused a 1.5-fold increase in secreted [(35)S]APP and a decrease in cell-associated [(35)S]APP. Although L-type voltage-dependent calcium channels (VDCC) have been implicated in A beta toxicity, the effect of L-type VDCC on APP secretion has not previously been examined. The L-type VDCC antagonists nifedipine and diltiazem both increased [(35)S]APP secretion into the medium but did not influence the effect of A beta on [(35)S]APP secretion. These studies suggest that A beta interferes with the secretory pathway of APP. Insofar as secreted APP has been proposed to have a neuroprotective function, the accumulation of A beta in the AD brain could decrease secreted APP and thereby indirectly increase A beta toxicity.

Substrate-bound beta-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures.

Accumulation of the beta-amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism of Abeta toxicity remains unclear. Abeta can bind to the extracellular matrix, a structure that regulates adhesive events such as neurite outgrowth and synaptogenesis. The binding of Abeta to the extracellular matrix suggests that Abeta may disrupt cell-substrate interactions. Therefore, the effect of substrate-bound Abeta on the growth of isolated chick sympathetic and mouse cortical neurons was examined. Abeta1-40 and Abeta1-42 had dose-dependent effects on cell morphology. When tissue culture plates were coated with 0.1-10 ng/well Abeta, neurite outgrowth increased. Higher amounts of Abeta peptides (> or =3 microg/well) inhibited outgrowth. The inhibitory effect was related to aggregation of the peptide, as preincubation of Abeta1-40 for 24 h at 37 degrees C (a process known to increase amyloid fibril formation) was necessary for inhibition of neurite outgrowth. Abeta29-42, but not Abeta1-28, also inhibited neurite outgrowth at high concentrations, demonstrating that the inhibitory domain is located within the hydrophobic C-terminal region. Abeta1-40, Abeta1-42, and Abeta29-42 also inhibited cell-substrate adhesion, indicating that the effect on neurite outgrowth may have been due to inhibition of cell adhesion. The results suggest that accumulation of Abeta may disrupt cell-adhesion mechanisms in vivo.

An unusually glycosylated form of acetylcholinesterase is a CSF biomarker for Alzheimer's disease.

The identification of a biochemical marker of Alzheimer's disease (AD) is a major research aim of many groups. Abnormal levels of tau and Abeta have been identified in the cerebrospinal fluid (CSF) of AD patients, although the sensitivity and specificity of the changes in these two biomarkers alone is not sufficient to be of diagnostic value. Recently, our group has identified an abnormality in the glycosylation of acetylcholinesterase (AChE). The increase in this glycoform of AChE is very specific for Alzheimer's disease and is not seen in many other neurological diseases including other dementias.

The amyloid beta-protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain-sensitive trypsin-like serine protease in cultures of chick sympathetic neurons.

The amyloid beta-protein precursor (APP) of Alzheimer's disease (AD) is cleaved either by alpha-secretase to generate an N-terminally secreted fragment, or by beta- and gamma-secretases to generate the beta-amyloid protein (Abeta). The accumulation of Abeta in the brain is an important step in the pathogenesis of AD. Alternative mRNA splicing can generate isoforms of APP which contain a Kunitz protease inhibitor (KPI) domain. However, little is known about the physiological function of this domain. In the present study, the metabolic turnover of APP was examined in cultured chick sympathetic neurons. APP was labelled by incubating neurons for 5 h with [35S]methionine and [35S]cysteine. Intracellular labelled APP decayed in a biphasic pattern suggesting that trafficking occurs through two metabolic compartments. The half-lives for APP in each compartment were 1.5 and 5.7 h, respectively. A small fraction (10%) of the total APP was secreted into the culture medium where it was degraded with a half-life of 9 h. Studies using specific protease inhibitors demonstrated that this extracellular breakdown was due to cleavage by a trypsin-like serine protease that was secreted into the culture medium. Significantly, this protease was inhibited by a recombinant isoform of APP (sAPP751), which contains a region homologous to the Kunitz protease inhibitor (KPI) domain. These results suggest that KPI forms of APP regulate extracellular cleavage of secreted APP by inhibiting the activity of a secreted APP-degrading protease.

Inhibition of neurite outgrowth from chick sympathetic neurons by cholinesterase inhibitors is not mediated by binding to cholinesterases.

Several studies have suggested a role for cholinesterases in regulating neurite outgrowth. Some acetylcholinesterase (AChE) inhibitors can inhibit neurite outgrowth, but it is unclear if this is due to inhibition of AChE. In this study, the effect of cholinesterase inhibitors on neurite outgrowth from chick sympathetic neurons was examined. Very high (micromolar) concentrations of tacrine and BW284c51 were needed to inhibit neurite outgrowth. In contrast, nanomolar concentrations were required to block cholinesterase activity. No correlation was found between the type of inhibitor or potency of cholinesterase inhibition and inhibition of neurite outgrowth. Both tacrine and BW284c51 were neurotoxic at concentrations that inhibited outgrowth. Therefore, the action of cholinesterase inhibitors on neurite outgrowth may be due to non-specific toxicity rather than to cholinesterase binding.

Apolipoprotein E promotes the binding and uptake of beta-amyloid into Chinese hamster ovary cells in an isoform-specific manner.

The epsilon4 allele of apolipoprotein E gene is a major risk factor for Alzheimer's disease. However, the mechanism by which the E4 isoform of apolipoprotein E increases the risk of Alzheimer's disease is poorly understood. To determine whether the isoform-specific effects of apolipoprotein E may be mediated via clearance of bound beta-amyloid, we examined the uptake of beta-amyloid 1-40 into Chinese hamster ovary cells in the presence or absence of the apolipoprotein E isoforms E2, E3 and E4. Apolipoprotein E2 and E3 treatments were associated with higher association of beta-amyloid with cells as compared to treatment with E4. Heparin blocked the association of beta-amyloid with cells, as did an antibody to one of the apolipoprotein E receptors (the low-density lipoprotein receptor-related protein). Thus, the apolipoproteins E2 and E3, but not E4, may play important roles in the clearance of beta-amyloid from the extracellular space via the low-density lipoprotein receptor-related protein.

Molecular isoform distribution and glycosylation of acetylcholinesterase are altered in brain and cerebrospinal fluid of patients with Alzheimer's disease.

The glycosylation of acetylcholinesterase (AChE) in CSF was analyzed by lectin binding. AChE from Alzheimer's disease (AD) patients was found to bind differently to two lectins, concanavalin A and wheat germ agglutinin, than AChE from controls. As multiple isoforms of AChE are present in both CSF and brain, we examined whether the abnormal glycosylation of AD AChE was due to changes in a specific molecular isoform. Globular amphiphilic dimeric (G2a) and monomeric (G1a) isoforms of AChE were found to be differentially glycosylated in AD CSF. Glycosylation of AChE was also altered in AD frontal cortex but not in cerebellum and was also associated with an increase in the proportion of light (G2 and G1) isoforms. This study demonstrates that the glycosylation of AChE is altered in the AD brain and that changes in AChE glycosylation in AD CSF may reflect changes in the distribution of brain isoforms. The study also suggests that glycosylation of AChE may be a useful diagnostic marker for AD.

Recombinant human amyloid precursor-like protein 2 (APLP2) expressed in the yeast Pichia pastoris can stimulate neurite outgrowth.

The human amyloid precursor-like protein 2 (APLP2) is a member of the Alzheimer's disease amyloid precursor protein (APP) gene family. The human APLP2 ectodomain (sAPLP2) was expressed in the yeast Pichia pastoris and the recombinant sAPLP2 was purified from the culture medium in a single step by metal-chelating Sepharose chromatography. The neuritotrophic activity of APLP2 was compared to the APP isoforms sAPP695 and sAPP751 on chick sympathetic neurones. APLP2 had neurite outgrowth-promoting activity similar to that of the APP isoforms. This suggests that APP and APLP2 have a similar or related role and supports the idea of a redundancy in function between the APP-gene family proteins.

Neurite-outgrowth regulating functions of the amyloid protein precursor of Alzheimer's disease.

Many studies have shown that breakdown of the amyloid protein precursor (APP) to produce the amyloid protein is an important step in the pathogenic mechanism which causes Alzheimer's disease (AD). However, little is known about the normal function of APP. Developmental studies show that APP expression increases during the period of brain development when neurite outgrowth and synaptogenesis is maximal. APP is expressed highly within growing neurites and in growth cones, and purified APP has been shown to stimulate neurite outgrowth from cells in culture. Thus APP may regulate neurite outgrowth or synaptogenesis in vivo. APP is actively secreted from many cells, and the C-terminally secreted APP has been shown to associate with components of the extracellular matrix, such as the heparan sulphate proteoglycans (HSPGs). Two putative heparin-binding domains on APP have been reported. Binding of HSPGs to an N-terminal heparin-binding domain (HBD-1) stimulates the effect of substrate-bound APP on neurite outgrowth. In the mature nervous system, APP may play an important role in the regulation of wound repair. It is highly likely that studies on the normal functions of APP will shed further light on aspects of the pathogenesis of AD.