Inhibition of Catalase Activity with 3-Amino-Triazole Enhances the Cytotoxicity of the Alzheimer’s Amyloid-β Peptide

Amyloid-β (Aβ) is a cytotoxic peptide implicated in the pathology of Alzheimer’s disease. The antioxidant enzyme catalase has been suggested to protect against Aβ cytotoxicity in both neuronal and non-neuronal cell types. Inhibition of endogenous catalase using 3-amino-1,2,4-triazole (3AT) in neuronal (NT-2) and myeloma (SP2/0-Ag-14) cell lines increases Aβ toxicity, suggesting that any protective role for endogenous catalase requires active enzyme. In Aβ treated myeloma cells there was a significant decrease in the total cell catalase activity and immunoreactivity. However, when the surviving live cell population was isolated following Aβ treatment the levels of catalase were significantly increased. The surviving live cell population from groups treated with both 3AT and Aβ contain elevated immunoreactive catalase levels suggesting that the protective role for endogenous catalase may have a component independent of the antioxidant activity, possibly by acting as an Aβ binding protein. Amyloid-β (Aβ) cytotoxicity can be prevented by Vitamin E treatment or an anti-Aβ monoclonal antibody (ALI01), both of which also prevent Aβ cytotoxicity in cells treated with 3AT. These observations suggest that Aβ mediated cell death in both neuronal and non-neuronal cells is mediated in part by actions to increase hydrogen peroxide. Catalase has a protective role, as a hydrogen peroxide-degrading enzyme and catalase inhibition by Aβ is not the direct cause of cytotoxicity.     

Deposition of Alzheimer’s vascular amyloid-β is associated with decreased expression of brain -3-hydroxyacyl-coenzyme A dehydrogenase (ERAB)

-3-hydroxyacyl-coenzyme A dehydrogenase type II (HADH) was described as an endoplasmic reticulum amyloid β-peptide-binding protein (ERAB), which enhances Aβ toxicity, and accumulates in neurons in Alzheimer’s disease (AD). Hence, HADH/ERAB was suggested to mediate the amyloid-induced neurodegeneration. We estimated the in vivo interactions of HADH and Aβ in an immunocytochemical study of ten Alzheimer’s disease and seven normal brains using five monoclonal HADH-specific antibodies. We found no HADH in amyloid plaques or vascular amyloid. The neuronal expression of HADH was not correlated with the severity of amyloid load in neuropil. HADH was expressed in vascular smooth muscle cells in young and old controls and in amyloid-free blood vessels in AD cases, but little or no HADH was in smooth muscle cells in arteries with amyloid deposits. The putative intracellular interaction between HADH and Aβ in amyloid-producing cells was further studied in vascular smooth muscle cells isolated from brain blood vessels with amyloid-β angiopathy — the cells that were shown previously to accumulate Aβ intracellularly [‘Research advances in Alzheimer’s disease and related disorders’ (1995) 747; Brain Res. 676 (1995) 225; Neurosci. Lett. 183 (1995) 120]. HADH had a mitochondrial localization and did not co-localize with an endoplasmic reticulum marker. Cells that accumulated Aβ were those with low expression of HADH and the proteins did not co-localize. Explanation of the association between low levels of HADH and deposition of Aβ by brain smooth muscle cells requires further studies.     

Amyloid-β: a chameleon walking in two worlds: a review of the trophic and toxic properties of amyloid-β

Although much maligned, the amyloid-β (Aβ) protein has been shown to possess a number of trophic properties that emanate from the protein’s ability to bind Cu, Fe and Zn. Aβ belongs to a group of proteins that capture redox metal ions (even under mildly acidotic conditions), thereby preventing them from participating in redox cycling with other ligands. The coordination of Cu appears to be crucial for Aβ’s own antioxidant activity that has been demonstrated both in vitro as well as in the brain, cerebrospinal fluid and plasma. The chelation of Cu by Aβ would therefore be predicted to dampen oxidative stress in the mildly acidotic and oxidative environment that accompanies acute brain trauma and Alzheimer’s disease (AD). Given that oxidative stress promotes Aβ generation, the formation of diffuse amyloid plaques is likely to be a compensatory response to remove reactive oxygen species. This review weighs up the evidence supporting both the trophic and toxic properties of Aβ, and while evidence for direct Aβ neurotoxicity in vivo is scarce, we postulate that the product of Aβ’s antioxidant activity, hydrogen peroxide (H₂O₂), is likely to mediate toxicity as the levels of this oxidant rise with the accumulation of Aβ in the AD brain. We propose that metal ion chelators, antioxidants, antiinflammatories and amyloid-lowering drugs that target the reduction of H₂O₂ and/or Aβ generation may be efficacious in decreasing neurotoxicity. However, given the antioxidant activity of Aβ, we suggest that the excessive removal of Aβ may prevent adequate chelation of metal ions and removal of O₂⁻, leading to enhanced, rather than reduced, neuronal oxidative stress.     

Platelet secretion of β-amyloid is increased in hypercholesterolaemia

Tissue accumulation of the cytotoxic β-amyloid peptide (Aβ) occurs in Alzheimer’s disease (AD), one possible source being the platelet. AD and cardiovascular disease may share some risk factors, including hypercholesterolaemia which is associated with increased platelet activity. We examined platelet Aβ release under resting and collagen-stimulated conditions in normocholesterolaemic and hypercholesterolaemic individuals. Resting platelet Aβ efflux was greater in hypercholesterolaemics than in normocholesterolaemics. Collagen-stimulated Aβ release was concentration-dependent and increased in hypercholesterolaemics. Resting Aβ release correlated positively with plasma total cholesterol and low-density lipoprotein (LDL) cholesterol, and inversely with platelet count. These data indicate that abnormal platelet Aβ release occurs in hypercholesterolaemia.     

Immunohistochemical localization of neprilysin in the human cerebral cortex: inverse association with vulnerability to amyloid β-protein (Aβ) deposition

We investigated the immunohistochemical localization of neprilysin, a putative amyloid β-protein (Aβ)-degrading enzyme, in postmortem human brain tissues. In the cerebral cortex, neprilysin immunoreactivity was weak, but relatively dense distribution was found in the primary somatosensory and visual cortices compared with the hippocampus and association cortices. In Alzheimer brain, neprilysin-positive dystrophic neurites occurred in senile plaques in the primary cortices, an observation that supports the relative abundance of neprilysin-positive neuronal processes. A paucity of neprilysin in the hippocampus and association cortices may contribute to the vulnerability of these areas to Aβ deposition.     

Testosterone attenuates β-amyloid toxicity in cultured hippocampal neurons

Accumulating evidence suggests that testosterone has neurotrophic and perhaps neuroprotective actions. Thus, age-related depletion of testosterone may increase the brain’s vulnerability to Alzheimer’s disease and related disorders. To begin investigating this issue, cultured neurons were exposed to the Alzheimer-related insult β-amyloid in the presence of testosterone. β-Amyloid neurotoxicity was significantly reduced by testosterone via a rapid, estrogen-independent mechanism. These data may provide additional insight into the treatment of age-related neurodegenerative disorders.     

The Ginkgo biloba extract EGb 761 rescues the PC12 neuronal cells from β-amyloid-induced cell death by inhibiting the formation of β-amyloid-derived diffusible neurotoxic ligands

β Amyloid (Aβ) treatment induced free radical production and increased glucose uptake, apoptosis and cell death in PC12 nerve cells. Addition of the standardized extract of Ginkgo biloba leaves, EGb 761 together with the Aβ protein prevented, in a dose-dependent manner, the Aβ-induced free radical production, increased glucose uptake, apoptosis and cell death. However, pretreatment of the cells with EGb 761 did not rescue the cells from the Aβ-induced toxicity although it prevented the Aβ-induced reactive oxygen species generation. Moreover, the terpene and flavonoid-free EGb 761 extract, HE 208, although inhibited the Aβ-induced increased glucose uptake, it failed to protect the cells from apoptosis and cytotoxicity induced by Aβ. In conclusion, these results indicate that the terpenoid and flavonoid constituents of EGb 761, acting probably in combination with components present in HE 208, are responsible for rescuing the neuronal cells from Aβ-induced apoptosis and cell death; their mechanism of action being distinct of their antioxidant properties. Because pre- and post-treatment with EGb 761 did not protect the cells from Aβ-induced neurotoxicity, we examined whether EGb 761 interacts directly with Aβ. Indeed, in vitro reconstitution studies demonstrated that EGb 761 inhibits, in a dose-dependent manner, the formation of β-amyloid-derived diffusible neurotoxic soluble ligands (ADDLs), suggested to be involved in the pathogenesis of Alzheimer’s disease.     

Inhibition of amyloid-beta-induced cell death in human brain pericytes in vitro

Amyloid-beta protein (A beta) deposition in the cerebral vascular walls is one of the key features of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). A beta(1-40) carrying the 'Dutch' mutation (HCHWA-D A beta(1-40)) induces pronounced degeneration of cultured human brain pericytes. In this study, we aimed to identify inhibitors of A beta-induced toxicity in human brain pericytes. The toxic effect of HCHWA-D A beta(1-40) on human brain pericytes was inhibited by co-incubation with catalase, but not with superoxide dismutase, glutathione or vitamin E analogue Trolox. Catalase interacts with A beta, both in cell cultures and in cell-free assays, and has a prominent effect on the amount and conformational state of A beta binding to the cell surface of human brain pericytes. This activity of catalase is likely based on its ability to bind and slowly degrade A beta and not by its usual capacity to convert hydrogen peroxide. Our data confirm that assembly of A beta at the cell surface of human brain pericytes is a crucial step in A beta-induced cellular degeneration of human brain pericytes. Inhibition of fibril formation at the cell surface could be an important factor in therapy aimed at reducing cerebral amyloid angiopathy.     

Reduction in somatostatin and substance P levels and choline acetyltransferase activity in the cortex and hippocampus of the rat after chronic intracerebroventricular infusion of β-amyloid (1-40)

The present study investigated the neurochemical and behavioural sequelae following chronic intracerebroventricular infusion of β-amyloid (1-40) in rats. β-amyloid was either infused intermittently via implanted cannulae on the day of operation and subsequently on postsurgical days 4, 7, 10, and 13 (Experiment 1), or continuously using osmotic pumps for 14 days (Experiment 2). The same amount of β-amyloid was delivered under both infusion regimes. In both experiments, β-amyloid infusion led to severe deficits in the acquisition of a spatial reference memory task conducted on postoperative days 10 to 14. The animals were sacrificed on the postoperative day 15 for neurochemical analyses. These included radioenzymatic and radioimmunoassays, designed to determine choline acetyltransferase activity and the contents of neuropeptides (somatostatin, substance P, and neuropeptide Y), respectively. Experiment 2 also included solution-hybridisation-RNAase protection assay for preprosomatostatin mRNA quantification. There was a significant reduction in choline acetyltransferase activity and in the levels of substance P as well as somatostatin and preprosomatostatin mRNA in the cortical mantle of β-amyloid-treated rats, compared to controls in both experiments. Appreciable reductions in choline acetyltransferase activity and somatostatin level were also apparent in the hippocampus. In contrast, β-amyloid infusion did not significantly affect the brain level of neuropeptide Y. The present study demonstrated that chronic infusion of β-amyloid can lead to a reduction in the levels of selected neuropeptides resembling the pattern seen in Alzheimer’s disease patients.     

A novel compound,RS-1178, specifically inhibits neuronal cell death mediated by beta-amyloid-induced macrophage activation in vitro

beta-Amyloid peptide (Abeta), a major component of senile plaques, the formation of which is characteristic of Alzheimer's disease (AD), is believed to induce inflammation in the brain leading to cell loss and cognitive decline. Accumulating evidence shows Abeta activates microglia, which play the role of the brain's immune system, and mediates inflammatory responses in the brain. Thus, a compound inhibiting Abeta-induced activation of microglia may lead to a novel therapy for AD. However, the compound should not inhibit natural immune responses during events such as bacterial infections. We investigated the effect of a synthesized compound, 7,8-dihydro-5-methyl-8-(1-phenylethyl)-6H-pyrrolo [3,2-e] [1,2,4] triazolo [1,5-a] pyrimidine (RS-1178) on macrophage activation induced by various stimulants. The activation of macrophages was determined by nitric oxide or tumor necrosis factor alpha production. RS-1178 inhibited Abeta-induced macrophage activation but did not inhibit zymosan A- nor lipopolysaccharide (LPS)-induced macrophage activation. Moreover, RS-1178 attenuated neurotoxicity due to Abeta-induced macrophage activation in neuron-macrophage co-cultures but not neurotoxicity due to zymosan A- or LPS-induced macrophage activation. In conclusion, RS-1178 showed a specific inhibitory effect on Abeta-induced macrophage activation. Although the exact mechanisms of this effect remain unknown, RS-1178 may provide a novel therapy for AD.     

Apolipoprotein E deposition and astrogliosis are associated with maturation of β-amyloid plaques in βAPPswe transgenic mouse: Implications for the pathogenesis of Alzheimer’s disease

A transgenic mouse expressing the human β-amyloid precursor protein with the ‘Swedish’ mutation, Tg2576, was used to investigate the mechanism of β-amyloid (Aβ) deposition. Previously, we have reported that the major species of Aβ in the amyloid plaques of Tg2576 mice are Aβ_1-40 and Aβ_1-42. Moreover, Aβ_1-42 deposition precedes Aβ_1-40 deposition, while Aβ_1-40 accumulates in the central part of the plaques later in the pathogenic process. Those data indicate that Aβ deposits in Tg2576 mice have similar characteristics to those in Alzheimer’s disease. In the present study, to understand more fully the amyloid deposition mechanism implicating Alzheimer’s disease pathogenesis, we examined immunohistochemically the distributions of apolipoprotein E (apoE) and Aβ in amyloid plaques of aged Tg2576 mouse brains. Our findings suggest that Aβ_1-42 deposition precedes apoE deposition, and that Aβ_1-40 deposition follows apoE deposition during plaque maturation. We next examined the relationship between apoE and astrogliosis associated with amyloid plaques using a double-immunofluorescence method. Extracellular apoE deposits were always associated with reactive astrocytes whose processes showed enhancement of apoE-immunoreactivity. Taken together, the characteristics of amyloid plaques in Tg2576 mice are similar to those in Alzheimer’s disease with respect to apoE and astrogliosis. Furthermore, apoE deposition and astrogliosis may be necessary for amyloid plaque maturation.     

β‐Catenin expression in human neural cell lines following exposure to cytokines and growth factors

β‐Catenin acts as a key mediator of the Wnt/Wingless signaling pathway involved in cell proliferation, differentiation and survival. Recent studies have shown that an unstable interaction between β‐catenin and the mutant presenilin‐1 induces neuronal apoptosis, and that β‐catenin levels are decreased in the brains of patients with Alzheimer’s disease (AD). Since activated microglia and astrocytes play a role in the process of neuronal degeneration in AD, the cytokine/growth factor‐regulated expression of β‐catenin in human neural cell lines, including NTera2 teratocarcinoma‐derived differentiated neurons (NTera2‐N), IMR‐32 neuroblastoma, SKN‐SH neuroblastoma and U‐373MG astrocytoma, was studied quantitatively following exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), brain‐derived neurotrophic factor (BDNF), tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1β, IL‐6, interferon (IFN)‐γ, transforming growth factor (TGF)‐β1, dibutyryl cyclic adenosine 3′,5′‐cyclic monophosphate (cAMP) (dbcAMP) or phorbol 12‐myristate 13‐acetate (PMA). β‐Catenin mRNA expressed constitutively in all of these cell lines was unaffected by treatment with any factors examined. In contrast, β‐catenin protein levels were reduced markedly in NTera2‐N cells by exposure to dbcAMP, EGF or bFGF, and in U‐373MG cells by treatment with dbcAMP or PMA, but were unaffected in any cell lines by BDNF, TNF‐α, IL‐1β, IL‐6, IFN‐γ or TGF‐β1. These results indicate that β‐catenin is expressed constitutively in human neural cells and downregulated at a protein level by a set of growth factors in a cell type‐specific manner.     

REVIEW: γ‐Secretase Inhibitors for the Treatment of Alzheimer's Disease: The Current State

Aims: Drugs currently used for the treatment of Alzheimer's disease (AD) partially stabilize patients’ symptoms without modifying disease progression. Brain accumulation of oligomeric species of β‐amyloid (Aβ) peptides, the principal components of senile plaques, is believed to play a crucial role in the development of AD. Based on this hypothesis, huge efforts are being spent to identify drugs able to interfere with proteases regulating Aβ formation from amyloid precursor protein (APP). This article briefly reviews the profile of γ‐secretase inhibitors, compounds that inhibit γ‐secretase, the pivotal enzyme that generates Aβ, and that have reached the clinic. Discussion: Several classes of potent γ‐secretase inhibitors have been designed and synthesized. Preclinical studies have indicated that these compounds are able to lower brain Aβ concentrations and, in some cases, reduce Aβ plaque deposition in transgenic mouse models of AD. The most developmentally advanced of these compounds is semagacestat, presently in Phase III clinical trials. In animals, semagacestat reduced Aβ levels in the plasma, cerebrospinal fluid (CSF), and the brain. However, studies have not reported on its cognitive effects. Studies in both healthy volunteers and patients with AD have demonstrated a dose‐dependent inhibition of plasma Aβ levels, and a recent study in healthy subjects demonstrated a robust, dose‐dependent inhibition of newly generated Aβ in the CSF after single oral doses. Conclusions: Unfortunately, γ‐secretase inhibitors may cause intestinal goblet cell hyperplasia, thymus atrophy, decrease in lymphocytes, and alterations in hair color, effects associated with the inhibition of the cleavage of Notch, a protein involved in cell development and differentiation. Nevertheless, at least other two promising γ‐secretase inhibitors are being tested clinically. This class of drugs represents a major hope to slow the rate of decline of AD.     

Up-regulation of E2F-1 in Down’s syndrome brain exhibiting neuropathological features of Alzheimer-type dementia

We studied the expression of the apoptosis-related protein, E2F-1, in Down’s syndrome (DS) brains. The immunoreactivity for E2F-1 was detected in the pyramidal neurons of the cerebral cortex from DS brains exhibiting the neuropathological features of dementia of Alzheimer type (DAT), in accordance with the amyloid β protein (Aβ) deposition in the neuron. Therefore, the implication is that Aβ deposition may trigger E2F-1-mediated neuronal apoptosis in DS brains with DAT.     

Sex differences in estrogen receptor α and β expression in vasopressin neurons of the supraoptic nucleus in elderly and Alzheimer’s disease patients: no relationship with cytoskeletal alterations

In various hypothalamic and adjacent brain regions we have previously found a remarkable increase in nuclear estrogen receptor staining in Alzheimer’s disease (AD). In order to see whether this was a general phenomenon or rather specific for those areas that are affected by the AD process we investigated ERα and ERβ expression in the arginine–vasopressin (AVP) neurons of the human dorsolateral suparoptic nucleus (dl-SON), that is the major source of plasma AVP. These neurons remain exceptionally intact in AD. Changes in ER expression were studied in relation to early Alzheimer changes (i.e. hyperphosphorylated tau) and neuronal metabolism in AD as determined by the size of the Golgi apparatus (GA) or cell size. No difference in neuronal metabolism (i.e. GA size or cell size) of AVP neurons was observed between AD and control patients and no early cytoskeletal AD alterations were found confirming the resistance of the dl-SON to AD. While no differences between AD and control patients were present for ERα and ERβ staining except for a lower proportion of nuclear ERβ AVP-positive neurons in AD subjects, complex sex differences not directly related to AD were observed within each group. The main finding of the present study is that in the dl-SON, that remains active and spared of AD changes, the increase in nuclear ERs seen in adjacent affected areas in AD patients does not occur. This indicates that a rise of nuclear ERs is not a generally occurring phenomenon but rather related to the pathogenetic alterations of the AD process.     

Reduction of cerebrospinal fluid amyloid β after systemic administration of M1 muscarinic agonists

Overproduction of the peptide amyloid β (Aβ) is a critical event in Alzheimer’s disease (AD). Systemic administration of 3 M1-selective muscarinic agonists, AF102B, AF150S and AF267B, decreased cerebrospinal fluid (CSF) Aβ concentrations; levels of CSF secreted β-APP were not significantly altered. Rabbits treated for 5 days with s.c. injections of each drug (2 mg/kg/day) had levels of CSF Aβ which were between 55 and 71% of control for Aβ 1–40 and between 59 and 84% of control for Aβ 1–42.     

Binding and uptake of Aβ1‐42 by primary human astrocytes in vitro

Clearance of the amyloid‐β peptide (Aβ) as a remedy for Alzheimer's disease (AD) is a major target in on‐going clinical trials. In vitro studies confirmed that Aβ is taken up by rodent astrocytes, but knowledge on human astrocyte‐mediated Aβ clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of Aβ1‐42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether α1‐antichymotrypsin (ACT), which is found in amyloid plaques and can influence Aβ fibrillogenesis, affects the Aβ uptake by human astrocytes. Upon over night exposure of astrocytes to FAM‐labeled Aβ1‐42 (10 μM) preparations, (80.7 ± 17.7)% fetal and (52.9 ± 20.9)% adult Aβ‐positive astrocytes (P = 0.018) were observed. No significant difference was found in Aβ1‐42 uptake between AD and non‐AD astrocytes, and no influence of ApoE genotype on Aβ1‐42 uptake was observed in any group. There was no difference in the percentage of Aβ‐positive cells upon exposure to Aβ1‐42 (10 μM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus Aβ1‐42 alone. CLSM revealed binding of Aβ1‐42 to the cellular surfaces and cellular internalization of smaller Aβ1‐42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte‐chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize Aβ1‐42, and fetal astrocytes were more efficient in Aβ1‐42 uptake than adult astrocytes. © 2008 Wiley‐Liss, Inc.     

Antiamnesic effects of azaindolizinone derivative ZSET845 on impaired learning and decreased ChAT activity induced by amyloid-β 25–35 in the rat

Antiamnesic effects of a newly synthesized azaindolizinone derivative ZSET845 were assessed in rats made learning ability deficient by amyloid-beta (Abeta)25-35 treatment. Intracerebroventricular injection of Abeta25-35 induced a marked decrease in step-through latency in passive avoidance task and reduction in choline acetyltransferase (ChAT) activity in the medial septum and hippocampus, but not in the basal forebrain and cortex. The number of ChAT-immunoreactive cells was decreased in the medial septum. Oral administration of ZSET845 at a dose of 1 or 10 mg/kg ameliorated learning impairment in passive avoidance task and enhanced ChAT activity in the basal forebrain, medial septum and hippocampus, and increased in the number of ChAT-immunoreactive cells in the medial septum in Abeta-treated rats to the levels of vehicle-injected control rats. These results suggest that ZSET845 is worth testing for further preclinical study aimed for the treatment of senile dementia such as Alzheimer's disease.     

Hippocampal estrogen β-receptor immunoreactivity is increased in Alzheimer’s disease

Post-menopausal estrogen use reduces the risk and severity of Alzheimer’s disease (AD). The present study investigates the distribution of both estrogen receptors ERα and ERβ in the human hippocampus in aged controls and in AD cases with immunohistochemistry. No ERα immunoreactivity was observed both in controls and in AD cases. On the other hand, ERβ was observed in some neuronal cells in the hippocampal subfields CA1–4, in astrocytes and in extracellular deposits both in controls and AD cases. The ERβ immunoreactivity was distinctly increased in all AD cases in cellular and extracellular localizations indicating a role for ERβ-mediated estrogen effects in AD-related neuropathology. This study provides the first demonstration of ERβ in human hippocampus in aged controls compared to AD cases.