The Luminescent Conjugated Oligothiophene h-FTAA Attenuates the Toxicity of Different Aß Species.

The prevailing opinion is that prefibrillar ß-amyloid (Aß) species, rather than end-stage amyloid fibrils, cause neuronal dysfunction in Alzheimer's disease, although the mechanisms behind Aß neurotoxicity remain to be elucidated. Luminescent conjugated oligothiophenes (LCOs) exhibit spectral properties upon binding to amyloid proteins and have previously been reported to change the toxicity of Aß1-42 and prion protein. In a previous study, we showed that an LCO, pentamer formyl thiophene acetic acid (p-FTAA), changed the toxicity of Aß1-42. Here we investigated whether an LCO, heptamer formyl thiophene acetic acid (h-FTAA), could change the toxicity of Aß1-42 by comparing its behavior with that of p-FTAA. Moreover, we investigated the effects on toxicity when Aß with the Arctic mutation (AßArc) was aggregated with both LCOs. Cell viability assays on SH-SY5Y neuroblastoma cells demonstrated that h-FTAA has a stronger impact on Aß1-42 toxicity than does p-FTAA. Interestingly, h-FTAA, but not p-FTAA, rescued the AßArc-mediated toxicity. Aggregation kinetics and binding assay experiments with Aß1-42 and AßArc when aggregated with both LCOs showed that h-FTAA and p-FTAA either interact with different species or affect the aggregation in different ways. In conclusion, h-FTAA protects against Aß1-42 and AßArc toxicity, thus showing h-FTAA to be a useful tool for improving our understanding of the process of Aß aggregation linked to cytotoxicity.

Amyloid-ß induced membrane damage instigates tunneling nanotube-like conduits by p21-activated kinase dependent actin remodulation.

Alzheimer's disease (AD) pathology progresses gradually via anatomically connected brain regions. Direct transfer of amyloid-ß1-42 oligomers (oAß) between connected neurons has been shown, however, the mechanism is not fully revealed. We observed formation of oAß induced tunneling nanotubes (TNTs)-like nanoscaled f-actin containing membrane conduits, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that oAß propagate from one cell to another via TNT-like structures. Preceding the formation of TNT-like conduits, we detected oAß-induced plasma membrane (PM) damage and calcium-dependent repair through lysosomal-exocytosis, followed by massive endocytosis to re-establish the PM. Massive endocytosis was monitored by an influx of the membrane-staining dye TMA-DPH and PM damage was quantified by propidium iodide influx in the absence of Ca2+. The massive endocytosis eventually caused accumulation of internalized oAß in Lamp1 positive multivesicular bodies/lysosomes via the actin cytoskeleton remodulating p21-activated kinase1 (PAK1) dependent endocytic pathway. Three-dimensional quantitative confocal imaging, structured illumination superresolution microscopy, and flowcytometry quantifications revealed that oAß induces activation of phospho-PAK1, which modulates the formation of long stretched f-actin extensions between cells. Moreover, the formation of TNT-like conduits was inhibited by preventing PAK1-dependent internalization of oAß using the small-molecule inhibitor IPA-3, a highly selective cell-permeable auto-regulatory inhibitor of PAK1. The present study reveals that the TNT-like conduits are probably instigated as a consequence of oAß induced PM damage and repair process, followed by PAK1 dependent endocytosis and actin remodeling, probably to maintain cell surface expansion and/or membrane tension in equilibrium.

Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the Aß fly model and the AßPP-BACE1 fly model. In the Aß fly model, the Aß peptide is fused to a secretion sequence and directly overexpressed. In the AßPP-BACE1 model, human AßPP and human BACE1 are expressed in the fly, resulting in in vivo production of Aß peptides and other AßPP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the Aß fly model, this correlates with high Aß1-42 levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the AßPP-BACE1 fly model, neuronal cell death correlates with low Aß1-42 levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of AßPP/Aß antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the AßPP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments.

Beneficial effects of increased lysozyme levels in Alzheimer's disease modelled in Drosophila melanogaster.

Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aß1-42 or AßPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aß1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aß1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aß1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aß1-42 , which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.

AßPP processing results in greater toxicity per amount of Aß1-42 than individually expressed and secreted Aß1-42 in Drosophila melanogaster.

The aggregation of the amyloid-ß (Aß) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimer's disease (AD). Aß peptides are generated from proteolytic processing of the transmembrane Aß precursor protein (AßPP) via sequential proteolysis through the ß-secretase activity of ß-site AßPP-cleaving enzyme (BACE1) and by the intramembranous enzyme γ-secretase. For over a decade, Drosophila melanogaster has been used as a model organism to study AD, and two different approaches have been developed to investigate the toxicity caused by AD-associated gene products in vivo In one model, the Aß peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human AßPP is co-expressed with human BACE1, resulting in production of the Aß peptide through the processing of AßPP by BACE1 and by endogenous fly γ-secretase. Here, we performed a parallel study of flies that expressed the Aß1-42 peptide alone or that co-expressed AßPP and BACE1. Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the Aß1-42, Aß1-40 and Aß1-38 peptides were examined. Our data reveal that the toxic effect per amount of detected Aß1-42 peptide was higher in the flies co-expressing AßPP and BACE1 than in the Aß1-42-expressing flies, and that the co-existence of Aß1-42 and Aß1-40 in the flies co-expressing AßPP and BACE1 could be of significant importance to the neurotoxic effect detected in these flies. Thus, the toxicity detected in these two fly models seems to have different modes of action and is highly dependent on how and where the peptide is generated rather than on the actual level of the Aß1-42 peptide in the flies. This is important knowledge that needs to be taken into consideration when using Drosophila models to investigate disease mechanisms or therapeutic strategies in AD research.

Distinct Lysosomal Network Protein Profiles in Parkinsonian Syndrome Cerebrospinal Fluid.

BACKGROUND: Clinical diagnosis of parkinsonian syndromes like Parkinson's disease (PD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) is hampered by overlapping symptomatology and lack of diagnostic biomarkers, and definitive diagnosis is only possible post-mortem. OBJECTIVE: Since impaired protein degradation plays an important role in many neurodegenerative disorders, we hypothesized that profiles of select lysosomal network proteins in cerebrospinal fluid could be differentially expressed in these parkinsonian syndromes. METHODS: Cerebrospinal fluid samples were collected from PD patients (n = 18), clinically diagnosed 4-repeat tauopathy patients; corticobasal syndrome (CBS) (n = 3) and PSP (n = 8); and pathologically diagnosed PSP (n = 8) and CBD patients (n = 7). Each patient set was compared to its appropriate control group consisting of age and gender matched individuals. Select lysosomal network protein levels were detected via Western blotting. Factor analysis was used to test the diagnostic sensitivity, specificity and accuracy of the select lysosomal network protein expression profiles. RESULTS: PD, CBD and PSP were markedly different in their cerebrospinal fluid lysosomal network protein profiles. Lysosomal-associated membrane proteins 1 and 2 were significantly decreased in PD; early endosomal antigen 1 was decreased and lysozyme increased in PSP; and lysosomal-associated membrane proteins 1 and 2, microtubule-associated protein 1 light chain 3 and lysozyme were increased in CBD. A panel of lysosomal-associated membrane protein 2, lysozyme and microtubule-associated protein 1 light chain discriminated between controls, PD and 4-repeat tauopathies. CONCLUSIONS: This study offers proof of concept that select lysosomal network proteins are differentially expressed in cerebrospinal fluid of Parkinson's disease, corticobasal syndrome and progressive supranuclear palsy. Lysosomal network protein analysis could be further developed as a diagnostic fluid biomarker in parkinsonian syndromes.

The Luminescent Oligothiophene p-FTAA Converts Toxic Aß1-42 Species into Nontoxic Amyloid Fibers with Altered Properties.

Aggregation of the amyloid-ß peptide (Aß) in the brain leads to the formation of extracellular amyloid plaques, which is one of the pathological hallmarks of Alzheimer disease (AD). It is a general hypothesis that soluble prefibrillar assemblies of the Aß peptide, rather than mature amyloid fibrils, cause neuronal dysfunction and memory impairment in AD. Thus, reducing the level of these prefibrillar species by using molecules that can interfere with the Aß fibrillation pathway may be a valid approach to reduce Aß cytotoxicity. Luminescent-conjugated oligothiophenes (LCOs) have amyloid binding properties and spectral properties that differ when they bind to protein aggregates with different morphologies and can therefore be used to visualize protein aggregates. In this study, cell toxicity experiments and biophysical studies demonstrated that the LCO p-FTAA was able to reduce the pool of soluble toxic Aß species in favor of the formation of larger insoluble nontoxic amyloid fibrils, there by counteracting Aß-mediated cytotoxicity. Moreover, p-FTAA bound to early formed Aß species and induced a rapid formation of ß-sheet structures. These p-FTAA generated amyloid fibrils were less hydrophobic and more resistant to proteolysis by proteinase K. In summary, our data show that p-FTAA promoted the formation of insoluble and stable Aß species that were nontoxic which indicates that p-FTAA might have therapeutic potential.

Protective properties of lysozyme on ß-amyloid pathology: implications for Alzheimer disease.

The hallmarks of Alzheimer disease are amyloid-ß plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-ß1-40 in vitro. In this study we found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localized with amyloid-ß in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-ß1-42 reduced the formation of soluble and insoluble amyloid-ß species, prolonged survival and improved the activity of amyloid-ß1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-ß increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-ß1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-ß species. Overall, these studies establish a protective role for lysozyme against amyloid-ß associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease.

A voltage dependent non-inactivating Na+ channel activated during apoptosis in Xenopus oocytes.

Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na(+) current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na(+)-channel blockers tetrodotoxin (1 µM) and amiloride (10 µM), while the Ca(2+)-channel blocker verapamil (50 µM) in the bath solution completely blocked the current. The intracellular Na(+) concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na(+) with either K(+) or Choline(+). Prevention of this influx of Na(+) also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na(+) increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na(+) channel is up-regulated during apoptosis and that influx of Na(+) is a crucial step in the apoptotic process in Xenopus oocytes.

Proteasome inhibition induces stress kinase dependent transport deficits--implications for Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, ß-amyloid and hyperphosphorylated tau. Both cellular systems responsible for clearance of misfolded and aggregated proteins, the lysosomal and the proteasomal, have been shown to be malfunctioning in the aged brain and more so in patients with neurodegenerative diseases, including AD. This malfunction could be contributing to ß-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated the impact of decreased proteasome activity on tau phosphorylation as well as on microtubule stability and transport. To do this, we used our recently developed neuronal model where human SH-SY5Y cells obtain neuronal morphology and function through differentiation. We found that exposure to low doses of the proteasome inhibitor MG-115 caused tau phosphorylation, microtubule destabilization and disturbed neuritic transport. Furthermore, reduced proteasome activity activated several proteins implicated in tau phosphorylation and AD pathology, including c-Jun N-terminal kinase, c-Jun and extracellular signal-regulated protein kinase (ERK) 1/2. Restoration of the microtubule transport was achieved by inhibiting ERK 1/2 activation, and simultaneous inhibition of both ERK 1/2 and c-Jun reversed the proteasome inhibition-induced tau phosphorylation. Taken together, this study suggests that a decrease in proteasome activity can, through activation of c-Jun and ERK 1/2, result in several events related to neurodegenerative diseases. Restoration of proteasome activity or modulation of ERK 1/2 and c-Jun function can open new treatment possibilities against neurodegenerative diseases such as AD.

Lysosomal network proteins as potential novel CSF biomarkers for Alzheimer's disease.

The success of future intervention strategies for Alzheimer's disease (AD) will likely rely on the development of treatments starting early in the disease course, before irreversible brain damage occurs. The pre-symptomatic stage of AD occurs at least one decade before the clinical onset, highlighting the need for validated biomarkers that reflect this early period. Reliable biomarkers for AD are also needed in research and clinics for diagnosis, patient stratification, clinical trials, monitoring of disease progression and the development of new treatments. Changes in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are among the first alterations observed in an AD brain. In this study, we performed a targeted search for lysosomal network proteins in human cerebrospinal fluid (CSF). Thirty-four proteins were investigated, and six of them, early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were significantly increased in the CSF from AD patients compared with neurological controls. These results were confirmed in a validation cohort of CSF samples, and patients with no neurochemical evidence of AD, apart from increased total-tau, were found to have EEA1 levels corresponding to the increased total-tau levels. These findings indicate that increased levels of LAMP-1, LAMP-2, LC3, Rab3 and Rab7 in the CSF might be specific for AD, and increased EEA1 levels may be a sign of general neurodegeneration. These six lysosomal network proteins are potential AD biomarkers and may be used to investigate lysosomal involvement in AD pathogenesis.

Alpha-synuclein biology in Lewy body diseases.

α-Synuclein is an abundantly expressed neuronal protein that is at the center of focus in understanding a group of neurodegenerative disorders called α-synucleinopathies, which are characterized by the presence of aggregated α-synuclein intracellularly. Primary α-synucleinopathies include Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, with α-synuclein also found secondarily in a number of other diseases, including Alzheimer's disease. Understanding how α-synuclein aggregates form in these different disorders is important for the understanding of its pathogenesis in Lewy body diseases. PD is the most prevalent of the α-synucleinopathies and much of the initial research on α-synuclein Lewy body pathology was based on PD but is also relevant to Lewy bodies in other diseases (dementia with Lewy bodies and Alzheimer's disease). Polymorphism and mutation studies of SNCA, the gene that encodes α-synuclein, provide much evidence for a causal link between α-synuclein and PD. Among the primary α-synucleinopathies, multiple system atrophy is unique in that α-synuclein deposition occurs in oligodendrocytes rather than neurons. It is unclear whether α-synuclein originates from oligodendrocytes or whether it is transmitted somehow from neurons. α-Synuclein exists as a natively unfolded monomer in the cytosol, but in the presence of lipid membranes it is thought to undergo a conformational change to a folded α-helical secondary structure that is prone to forming dimers and oligomers. Posttranslational modification of α-synuclein, such as phosphorylation, ubiquitination and nitration, has been widely implicated in α-synuclein aggregation process and neurotoxicity. Recent studies using animal and cell models, as well as autopsy studies of patients with neuron transplants, provided compelling evidence for prion-like propagation of α-synuclein. This observation has implications for therapeutic strategies, and much recent effort is focused on developing antibodies that target extracellular α-synuclein.

The lysosome: from waste bag to potential therapeutic target.

Lysosomes are ubiquitous membrane-bound intracellular organelles with an acidic interior. They are central for degradation and recycling of macromolecules delivered by endocytosis, phagocytosis, and autophagy. In contrast to the rather simplified view of lysosomes as waste bags, nowadays lysosomes are recognized as advanced organelles involved in many cellular processes and are considered crucial regulators of cell homeostasis. The function of lysosomes is critically dependent on soluble lysosomal hydrolases (e.g. cathepsins) as well as lysosomal membrane proteins (e.g. lysosome-associated membrane proteins). This review focuses on lysosomal involvement in digestion of intra- and extracellular material, plasma membrane repair, cholesterol homeostasis, and cell death. Regulation of lysosomal biogenesis and function via the transcription factor EB (TFEB) will also be discussed. In addition, lysosomal contribution to diseases, including lysosomal storage disorders, neurodegenerative disorders, cancer, and cardiovascular diseases, is presented.

Sensitivity to lysosome-dependent cell death is directly regulated by lysosomal cholesterol content.

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-ß-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

Lysosome-mediated apoptosis is associated with cathepsin D-specific processing of bid at Phe24, Trp48, and Phe183.

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Bax activator Bid, and translocation of Bax to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.

Amyloid-ß secretion, generation, and lysosomal sequestration in response to proteasome inhibition: involvement of autophagy.

The proteasome is important for degradation of worn out and misfolded proteins. Decreased proteasome activity has been implicated in Alzheimer's disease (AD). Proteasome inhibition induces autophagy, but it is still unknown whether autophagy is beneficial or deleterious to AD neurons, as the autophagosome has been suggested as a site of amyloid-ß (Aß) generation. In this study, we investigated the effect of proteasome inhibition on Aß accumulation and secretion, as well as the processing of amyloid-ß protein precursor (AßPP) in AßPP(Swe) transfected SH-SY5Y neuroblastoma cells. We show that proteasome inhibition resulted in autophagy-dependent accumulation of Aß in lysosomes, and increased levels of intracellular and secreted Aß. The enhanced levels of Aß could not be explained by increased amounts of AßPP. Instead, reduced degradation of the C-terminal fragment of AßPP (C99) by the proteasome makes C99 available for γ-secretase cleavage, leading to Aß generation. Inhibition of autophagy after proteasome inhibition led to reduced levels of intracellular, but not secreted Aß, and tended to further increase the C99 to AßPP ratio, supporting involvement of the autophagosome in Aß generation. Furthermore, proteasome inhibition caused a reduction in cellular viability, which was reverted by inhibition of autophagy. Dysfunction of the proteasome could cause lysosomal accumulation of Aß, as well as increased generation and secretion of Aß, which is partly facilitated by autophagy. As a decrease in cellular viability was also detected, it is possible that upregulation of autophagy is an unsuccessful rescue mechanism, which instead of being protective, contributes to AD pathogenesis.

Identification of distinct physiochemical properties of toxic prefibrillar species formed by Aß peptide variants.

The formation of amyloid-ß peptide (Aß) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of Aß. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of Aß-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aß aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those Aß peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the Aß peptide to form nontoxic versus toxic species.

Macroautophagy-generated increase of lysosomal amyloid ß-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells.

Increasing evidence suggests the toxicity of intracellular amyloid ß-protein (Aß) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aß within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aß monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aß production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aß resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aß accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.

Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation.

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.

Increased expression of the lysosomal cholesterol transporter NPC1 in Alzheimer's disease.

The Niemann-Pick type C1 (NPC1) protein mediates the trafficking of cholesterol from lysosomes to other organelles. Mutations in the NPC1 gene lead to the retention of cholesterol and other lipids in the lysosomal compartment, and such defects are the basis of NPC disease. Several parallels exist between NPC disease and Alzheimer's disease (AD), including altered cholesterol homeostasis, changes in the lysosomal system, neurofibrillary tangles, and increased amyloid-beta generation. How the expression of NPC1 in the human brain is affected in AD has not been investigated so far. In the present study, we measured NPC1 mRNA and protein expression in three distinct regions of the human brain, and we revealed that NPC1 expression is upregulated at both mRNA and protein levels in the hippocampus and frontal cortex of AD patients compared to control individuals. In the cerebellum, a brain region that is relatively spared in AD, no difference in NPC1 expression was detected. Similarly, murine NPC1 mRNA levels were increased in the hippocampus of 12-month-old transgenic mice expressing a familial AD form of human amyloid-beta precursor protein (APP) and presenilin-1 (APP/PS1tg) compared to 12-month-old wild type mice, whereas no change in NPC1 was detected in mouse cerebellum. Immunohistochemical analysis of human hippocampus indicated that NPC1 expression was strongest in neurons. However, in vitro studies revealed that NPC1 expression was not induced by transfecting SK-N-SH neurons with human APP or by treating them with oligomeric amyloid-beta peptide. Total cholesterol levels were reduced in hippocampus from AD patients compared to control individuals, and it is therefore possible that the increased expression of NPC1 is linked to perturbed cholesterol homeostasis in AD.