Beta-amyloid precursor polypeptide in SAMP8 mice affects learning and memory

Senescence accelerated (SAMP8 [P8]) mice develop age-related deficits in memory and learning. We show that increased expression of amyloid precursor protein (APP) and its mRNA in the hippocampus are also age-related. Immunocytochemical data suggest that a critical amount of APP expression may be needed to generate amyloid (Aβ) protein plaques in the hippocampus. Deficits in acquisition and retention test performance were alleviated by administration of antibody to Aβ protein into the cerebral ventricles. This reversal of cognitive deficits provides a link between increased expression of both APP and Aβ protein and learning and memory loss in these mice.     

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating ��-Secretase Complex Assembly and Dictating the Amyloid �� Peptide (A��) Profile without Affecting Notch Processing

γ-Secretase is an enzyme complex that mediates both Notch signaling and β-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid β peptide (Aβ), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of γ-secretase gives rise to Aβ peptides of different lengths, where Aβ42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320–374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of γ-secretase, for γ-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, γ-secretase complex formation, and had a differential effect on Aβ-peptide production. Although the production of Aβ38, Aβ39, and Aβ40 was severely impaired, the effect on Aβ42 was affected to a lesser extent, implying that the production of the AD-related Aβ42 peptide is separate from the production of the Aβ38, Aβ39, and Aβ40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ϵ/S3 and γ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Aβ peptides without affecting Notch processing, two parameters of significance when considering γ-secretase as a target for pharmaceutical intervention in AD.     

Passage of amyloid beta protein antibody across the blood-brain barrier in a mouse model of Alzheimer's disease

Vaccinations against amyloid β protein (AβP) reduce amyloid deposition and reverse learning and memory deficits in mouse models of Alzheimer’s disease. This has raised the question of whether circulating antibodies, normally restricted by the blood–brain barrier (BBB), can enter the brain [Nat. Med. 7 (2001) 369–372]. Here, we show that antibody directed against AβP does cross the BBB at a very low rate. Entry is by way of the extracellular pathways with about 0.11% of an intravenous (i.v.) dose entering the brain by 1 h. Clearance of antibody from brain increasingly dominates over time, but antibody is still detectable in brain 72 h after i.v. injection. Uptake and clearance is not altered in mice overexpressing AβP. This ability to enter and exit the brain even in the presence of increased brain ligand supports the use of antibody in the treatment of Alzheimer’s and other diseases of the brain.     

Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer's disease

In Alzheimer’s disease (AD), the neuropathologic hallmarks of β-amyloid deposition and neurofibrillary degeneration are associated with early and progressive pathology of the endosomal–lysosomal system. Abnormalities of autophagy, a major pathway to lysosomes for protein and organelle turnover, include marked accumulations of autophagy-related vesicular compartments (autophagic vacuoles or AVs) in affected neurons. Here, we investigated the possibility that AVs contain the proteases and substrates necessary to cleave the amyloid precursor protein (APP) to Aβ peptide that forms β-amyloid, a key pathogenic factor in AD. AVs were highly purified using a well-established metrizamide gradient procedure from livers of transgenic YAC mice overexpressing wild-type human APP. By Western blot analysis, AVs contained APP, βCTF - the β-cleaved carboxyl-terminal domain of APP, and BACE, the protease-mediating β-cleavage of APP. β-Secretase activity measured against a fluorogenic peptide was significantly enriched in the AV fraction relative to whole-liver lysate. Compared to other recovered subcellular fractions, AVs exhibited the highest specific activity of γ-secretase based on a fluorogenic assay and inhibition by a specific inhibitor of γ-secretase, DAPT. AVs were also the most enriched subcellular fraction in levels of the γ-secretase components presenilin and nicastrin. Immunoelectron microscopy demonstrated selective immunogold labeling of AVs with antibodies specific for the carboxyl termini of human Aβ40 and Aβ42. These data indicate that AVs are a previously unrecognized and potentially highly active compartment for Aβ generation and suggest that the abnormal accumulation of AVs in affected neurons of the AD brain contributes to β-amyloid deposition.     

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer''s disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer''s disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)¹, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology². The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.     

Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid ��-related Behavioral Deficits in a Mouse Model of Alzheimer Disease

Immunotherapy targeting of amyloid β (Aβ) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Aβ-targeted immunotherapy has also been demonstrated to reverse Aβ-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Aβ, Aβ_3–7, differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Aβ in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Aβ. The conformation of the Aβ peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Aβ:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Aβ species postulated to underlie cognitive deficits in AD.     

Alzheimer's Disease Related Markers,Cellular Toxicity and Behavioral Deficits Induced Six Weeks after Oligomeric Amyloid-β Peptide Injection in Rats

Alzheimer’s disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25–35) oligomers (oAβ_25–35) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ_25–35 and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ_25–35 was still present in the brain after 6 weeks. oAβ_25–35 injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ_25–35 injection resulted in increased APP expression, Aβ_1–42 generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.     

Zinc overload enhances APP cleavage and Aβ deposition in the Alzheimer mouse brain

Background

Abnormal zinc homeostasis is involved in β-amyloid (Aβ) plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer''s disease (AD). However, the involvement of zinc in amyloid precursor protein (APP) processing and Aβ deposition has not been well established in AD animal models in vivo.

Methodology/Principal Findings

In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aβ deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro.

Conclusions/Significance

The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.     

In vitro and in vivo degradation of Abeta peptide by peptidases coupled to erythrocytes

It is generally believed that amyloid β peptides (Aβ) are the key mediators of Alzheimer's disease. Therapeutic interventions have been directed toward impairing the synthesis or accelerating the clearance of Aβ. An equilibrium between blood and brain Aβ exists mediated by carriers that transport Aβ across the blood–brain barrier. Passive immunotherapy has been shown to be effective in mouse models of AD, where the plasma borne antibody binds plasma Aβ causing an efflux of Aβ from the brain. As an alternative to passive immunotherapy we have considered the use of Aβ-degrading peptidases to lower plasma Aβ levels. Here we compare the ability of three Aβ-degrading peptidases to degrade Aβ. Biotinylated peptidases were coupled to the surface of biotinylated erythrocytes via streptavidin. These erythrocyte-bound peptidases degrade Aβ peptide in plasma. Thus, peptidases bound to or expressed on the surface of erythroid cells represent an alternative to passive immunotherapy.     

Immunomodulation targeting abnormal protein conformation reduces pathology in a mouse model of Alzheimer's disease

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high β-sheet content and resistance to degradation. The most common is Alzheimer''s disease (AD) where the normal soluble amyloid β (sAβ) peptide is converted into highly toxic oligomeric Aβ and fibrillar Aβ that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAβ and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Aβ or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Aβ in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Aβ40/42 levels, as well as reduced Aβ oligomer levels. This type of immunomodulation has the potential to be a universal β-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases.     

Down-regulation of Seladin-1 Increases BACE1 Levels and Activity through Enhanced GGA3 Depletion during Apoptosis

Seladin-1 is a neuroprotective protein selectively down-regulated in brain regions affected in Alzheimer disease (AD). Seladin-1 protects cells against β-amyloid (Aβ) peptide 42- and oxidative stress-induced apoptosis activated by caspase-3, a key mediator of apoptosis. Here, we have employed RNA interference to assess the molecular effects of seladin-1 down-regulation on the β-secretase (BACE1) function and β-amyloid precursor protein (APP) processing in SH-SY5Y human neuroblastoma cells in both normal and apoptotic conditions. Our results show that ∼60% reduction in seladin-1 protein levels, resembling the decrease observed in AD brain, did not significantly affect APP processing or Aβ secretion in normal growth conditions. However, under apoptosis, seladin-1 small interfering RNA (siRNA)-transfected cells showed increased caspase-3 activity on average by 2-fold when compared with control siRNA-transfected cells. Increased caspase-3 activity coincided with a significant depletion of the BACE1-sorting protein, GGA3 (Golgi-localized γ-ear-containing ADP-ribosylation factor-binding protein), and subsequently augmented BACE1 protein levels and activity. Augmented BACE1 activity in turn correlated with the enhanced β-amyloidogenic processing of APP and ultimately increased Aβ production. These adverse changes associated with decreased cell viability in seladin-1 siRNA-transfected cells under apoptosis. No changes in GGA3 or BACE1 levels were found after seladin-1 knockdown in normal growth conditions. Collectively, our results suggest that under stress conditions, reduced seladin-1 expression results in enhanced GGA3 depletion, which further leads to augmented post-translational stabilization of BACE1 and increased β-amyloidogenic processing of APP. These mechanistic findings related to seladin-1 down-regulation are important in the context of AD as the oxidative stress-induced apoptosis plays a key role in the disease pathogenesis.     

Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease

Background

Alzheimer''s disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease.

Methodology/Principal Findings

We differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production.

Conclusions/Significance

These results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.     

Loss of Function of ATXN1 Increases Amyloid ��-Protein Levels by Potentiating ��-Secretase Processing of ��-Amyloid Precursor Protein

Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date ∼80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Aβ, the proteolytic product of β-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Aβ and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Aβ40 and Aβ42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Aβ levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Aβ levels is modulated via β-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating β-secretase cleavage of APP and Aβ levels.     

Prion Protein-mediated Toxicity of Amyloid-β Oligomers Requires Lipid Rafts and the Transmembrane LRP1

Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrP^C) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrP^C. The binding of Aβ oligomers to cell surface PrP^C, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrP^C, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrP^C impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrP^C-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrP^C in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD.     

Effects of heparin and enoxaparin on APP processing and Aβ production in primary cortical neurons from Tg2576 mice

Background

Alzheimer''s disease (AD) is caused by accumulation of Aβ, which is produced through sequential cleavage of β-amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE1) and γ-secretase. Enoxaparin, a low molecular weight form of the glycosaminoglycan (GAG) heparin, has been reported to lower Aβ plaque deposition and improve cognitive function in AD transgenic mice.

Methodology/Principal Findings

We examined whether heparin and enoxaparin influence APP processing and inhibit Aβ production in primary cortical cell cultures. Heparin and enoxaparin were incubated with primary cortical cells derived from Tg2576 mice, and the level of APP and proteolytic products of APP (sAPPα, C99, C83 and Aβ) was measured by western blotting. Treatment of the cells with heparin or enoxaparin had no significant effect on the level of total APP. However, both GAGs decreased the level of C99 and C83, and inhibited sAPPα and Aβ secretion. Heparin also decreased the level of β-secretase (BACE1) and α-secretase (ADAM10). In contrast, heparin had no effect on the level of ADAM17.

Conclusions/Significance

The data indicate that heparin and enoxaparin decrease APP processing via both α- and β-secretase pathways. The possibility that GAGs may be beneficial for the treatment of AD needs further study.     

��-Secretase Inhibitor Potency Is Decreased by Aberrant ��-Cleavage Location of the ��Swedish Mutant�� Amyloid Precursor Protein

The amyloid-β (Aβ) peptide, widely known as the causative molecule of Alzheimer disease (AD), is generated by the sequential cleavage of amyloid precursor protein (APP) by the aspartyl proteases BACE1/β-secretase and presenilin/γ-secretase. Inhibition of BACE1, therefore, is a promising strategy for preventing the progression of AD. However, β-secretase inhibitors (BSIs) exhibit unexpectedly low potency in cells expressing “Swedish mutant” APP (APPswe) and in the transgenic mouse Tg2576, an AD model overexpressing APPswe. The Swedish mutation dramatically accelerates β-cleavage of APP and hence the generation of Aβ; this acceleration has been assumed to underlie the poor inhibitory activity of BSI against APPswe processing. Here, we studied the mechanism by which the Swedish mutation causes this BSI potency decrease. Surprisingly, decreased BSI potency was not observed in an in vitro assay using purified BACE1 and substrates, indicating that the accelerated β-cleavage resulting from the Swedish mutation is not its underlying cause. By focusing on differences between the cell-based and in vitro assays, we have demonstrated here that the potency decrease is caused by the aberrant subcellular localization of APPswe processing and not by accelerated β-cleavage or the accumulation of the C-terminal fragment of β-cleaved APP. Because most patients with sporadic AD express wild type APP, our findings suggest that the wild type mouse is superior to the Tg2576 mouse as a model for determining the effective dose of BSI for AD patients. This work provides novel insights into the potency decrease of BSI and valuable suggestions for its development as a disease-modifying agent.     

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide

Background

The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer''s disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species.

Methodology/Principal Findings

To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species.

Conclusions

Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.     

The inhalation anesthetic desflurane induces caspase activation and increases amyloid beta-protein levels under hypoxic conditions

Perioperative factors including hypoxia, hypocapnia, and certain anesthetics have been suggested to contribute to Alzheimer disease (AD) neuropathogenesis. Desflurane is one of the most commonly used inhalation anesthetics. However, the effects of desflurane on AD neuropathogenesis have not been previously determined. Here, we set out to assess the effects of desflurane and hypoxia on caspase activation, amyloid precursor protein (APP) processing, and amyloid β-protein (Aβ) generation in H4 human neuroglioma cells (H4 naïve cells) as well as those overexpressing APP (H4-APP cells). Neither 12% desflurane nor hypoxia (18% O₂) alone affected caspase-3 activation, APP processing, and Aβ generation. However, treatment with a combination of 12% desflurane and hypoxia (18% O₂) (desflurane/hypoxia) for 6 h induced caspase-3 activation, altered APP processing, and increased Aβ generation in H4-APP cells. Desflurane/hypoxia also increased levels of β-site APP-cleaving enzyme in H4-APP cells. In addition, desflurane/hypoxia-induced Aβ generation could be reduced by the broad caspase inhibitor benzyloxycarbonyl-VAD. Finally, the Aβ aggregation inhibitor clioquinol and γ-secretase inhibitor L-685,458 attenuated caspase-3 activation induced by desflurane/hypoxia. In summary, desflurane can induce Aβ production and caspase activation, but only in the presence of hypoxia. Pending in vivo confirmation, these data may have profound implications for anesthesia care in elderly patients, and especially those with AD.An estimated 200 million patients worldwide undergo surgery each year. Several reports have suggested that anesthesia and surgery may facilitate development of Alzheimer disease (AD)⁴ (1–3). A recent study also reported that patients having coronary artery bypass graft surgery under general anesthesia are at increased risk for AD as compared with those having percutaneous transluminal coronary angioplasty under local anesthesia (4).Genetic evidence, confirmed by neuropathological and biochemical findings, indicates that excessive production and/or accumulation of amyloid β-protein (Aβ) play a fundamental role in the pathology of AD (reviewed in Refs. 5 and 6). Aβ is produced via serial proteolysis of amyloid precursor protein (APP) by aspartyl protease β-site APP-cleaving enzyme (BACE), or β-secretase, andγ-secretase. BACE cleaves APP to generate a 99-residue membrane-associated C terminus fragment (APP-C99). APP-C99 is further cleaved by γ-secretase to release 4-kDa Aβ and β-amyloid precursor protein intracellular domain (7–9). Presenilin and γ-secretase co-fractionate as a detergent-sensitive, high molecular weight complex (10) that includes at least three other proteins, nicastrin/APH-2, APH-1, and PEN-2, all of which are necessary and sufficient for γ-secretase activity (11–13). Increasing evidence indicates that apoptosis is associated with a variety of neurodegenerative disorders, including AD (Refs. 14–17; reviewed in Ref. 18). Aβ has been shown to cause caspase activation and apoptosis, which can in turn potentiate Aβ generation (16, 19–28). Finally, fibrillar aggregates of Aβ and oligomeric species of Aβ are more neurotoxic (29–37).Perioperative factors, including hypoxia (38–42), hypocapnia (43), and anesthetics (44–47), have been reported to potentially contribute to AD neuropathogenesis. These perioperative factors may also cause post-operative cognitive dysfunction, a dementia associated with surgery and anesthesia, by triggering AD neuropathogenesis.Isoflurane, sevoflurane, and desflurane are the most commonly used inhalation anesthetics. It has been reported that isoflurane enhances the oligomerization and cytotoxicity of Aβ (44) and induces apoptosis (48–51). Our recent studies have shown that a clinically relevant concentration of isoflurane can lead to caspase-3 activation, decrease cell viability, alter APP processing, and increase Aβ generation in human H4 neuroglioma cells overexpressing human APP (45–47). Loop et al. (49) reported that isoflurane and sevoflurane, but not desflurane, can induce caspase activation and apoptosis in human T lymphocytes. However, effects of desflurane and desflurane plus other perioperative risk factors, e.g. hypoxia, on APP processing and Aβ generation have not been assessed.In the present study, we set out to determine effects of desflurane, hypoxia, and the combination of the two (desflurane/hypoxia) on caspase-3 activation, APP processing, and Aβ generation in H4 human neuroglioma cells (H4 naïve cells) and H4 naïve cells stably transfected to express full-length (FL) APP (H4-APP cells). We also investigated whether the caspase inhibitor, Z-VAD, the γ-secretase inhibitor L-685,458, and the Aβ aggregation inhibitor clioquinol could attenuate desflurane/hypoxia-induced caspase-3 activation and Aβ generation.     

Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins

Background

Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ) of Alzheimer''s disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP).

Methodology/Principal Findings

Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y), whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes.

Conclusions/Significance

These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer''s disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.