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| 2. |
- Agholme, Lotta, et al.
(författare)
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Amyloid-β Secretion, Generation, and Lysosomal Sequestration in Response to Proteasome Inhibition : Involvement of Autophagy
- 2012
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Ingår i: Journal of Alzheimer's Disease. - : I O S Press. - 1387-2877 .- 1875-8908. ; 31:2, s. 343-358
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Tidskriftsartikel (refereegranskat)abstract
- 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-β (Aβ) generation. In this study, we investigated the effect of proteasome inhibition on Aβ accumulation and secretion, as well as the processing of amyloid-β protein precursor (AβPP) in AβPPSwe transfected SH-SY5Y neuroblastoma cells. We show that proteasome inhibition resulted in autophagy-dependent accumulation of Aβ in lysosomes, and increased levels of intracellular and secreted Aβ. The enhanced levels of Aβ could not be explained by increased amounts of AβPP. Instead, reduced degradation of the C-terminal fragment of AβPP (C99) by the proteasome makes C99 available for γ-secretase cleavage, leading to Aβ generation. Inhibition of autophagy after proteasome inhibition led to reduced levels of intracellular, but not secreted Aβ, and tended to further increase the C99 to AβPP ratio, supporting involvement of the autophagosome in Aβ 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 Aβ, as well as increased generation and secretion of Aβ, 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.
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| 3. |
- Agholme, Lotta, et al.
(författare)
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An In Vitro Model for Neuroscience: Differentiation of SH-SY5Y Cells into Cells with Morphological and Biochemical Characteristics of Mature Neurons
- 2010
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Ingår i: Journal of Alzheimer's Disease. - : Ios Press. - 1387-2877 .- 1875-8908. ; 20:4, s. 1069-1082
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Tidskriftsartikel (refereegranskat)abstract
- Neuroscience, including research on Alzheimers disease, is hampered by the lack of suitable in vitro models to study the human nervous system. To counteract this, many attempts to differentiate cell lines into more neuron-like cells have been performed, resulting in partial expression of neuronal features. Furthermore, it has been reported that neuroblastoma cell lines lack mature isoforms of tau. Our aim was to develop an improved in vitro model, generating sustainable cells with morphology and biochemistry of human, mature neurons. To obtain cells with neuronal differentiation and function, we investigated the effect of combining three-dimensional culturing of SH-SY5Y cells in extracellular matrix (ECM) gel with several factors reported to have neuro-differentiating effects. This resulted in cells with apparent neuronal morphology with long, extensively branched neurites. Further investigation revealed expression of several neurospecific markers including synapse protein Sv2 and nuclear marker NeuN, as well as the presence of synapses and axonal vesicle transport. In addition, these cells expressed mature tau isoforms, and tau protein expression was significantly increased compared to undifferentiated cells, reaching levels found in adult human brain. In conclusion, we found that pre-treatment with retinoic acid followed by ECM gel culturing in combination with brain derived neurotrophic factor, neuregulin beta(1), nerve growth factor, and vitamin D-3 treatment generated sustainable cells with unambiguous resemblance to adult neurons. These cells also expresses adult splicing forms of tau with neuronal localization, making this cellular in vitro model useful in many areas of neuroscience research, particularly the Alzheimers disease field.
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| 4. |
- Agholme, Lotta, et al.
(författare)
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Proteasome Inhibition Induces Stress Kinase Dependent Transport Deficits – Implications for Alzheimer’s Disease
- 2014
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Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 58, s. 29-39
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, β-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 AD patients. This malfunction could be the cause of β-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated how decreased proteasome activity affects AD related pathophysiological changes of microtubule transport and stability, as well as tau phosphorylation. 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 disturbed neuritic transport, together with microtubule destabilization and tau phosphorylation. Furthermore, reduced proteasome activity activated several kinases implicated in AD pathology, including JNK, c-Jun and 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 contributing to AD pathology. Restoring proteasome function or inhibiting ERK 1/2 and c-Jun could therefore be used as novel treatments against AD.
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| 5. |
- Appelqvist, Hanna, et al.
(författare)
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Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation
- 2011
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Ingår i: American Journal of Pathology. - : American Society for Investigative Pathology (ASIP). - 0002-9440 .- 1525-2191. ; 178:2, s. 629-639
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Tidskriftsartikel (refereegranskat)abstract
- 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 0-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.
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| 6. |
- Appelqvist, Hanna, et al.
(författare)
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Lysosome-Mediated Apoptosis is Associated with Cathepsin D-Specific Processing of Bid at Phe24,Trp48, and Phe183
- 2012
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Ingår i: Annals of Clinical and Laboratory Science. - : Institute for Clinical Science. - 0091-7370 .- 1550-8080. ; 42:3, s. 231-242
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Tidskriftsartikel (refereegranskat)abstract
- 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 Box activator Bid, and translocation of Box 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.
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| 7. |
- Appelqvist, Hanna, et al.
(författare)
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Sensitivity to Lysosome-Dependent Cell Death is Directly Regulated by Lysosomal Cholesterol Content
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:11
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Tidskriftsartikel (refereegranskat)abstract
- 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-β-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.
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| 8. |
- Armstrong, Andrea, et al.
(författare)
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Lysosomal Network Proteins as Potential Novel CSF Biomarkers for Alzheimers Disease
- 2014
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Ingår i: Neuromolecular medicine. - : Humana Press. - 1535-1084 .- 1559-1174. ; 16:1, s. 150-160
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Tidskriftsartikel (refereegranskat)abstract
- The success of future intervention strategies for Alzheimers 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.
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| 9. |
- Bergkvist, Liza, et al.
(författare)
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A beta PP processing results in greater toxicity per amount of A beta(1-42) than individually expressed and secreted A beta(1-42) in Drosophila melanogaster
- 2016
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Ingår i: BIOLOGY OPEN. - : COMPANY OF BIOLOGISTS LTD. - 2046-6390. ; 5:8, s. 1030-1039
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of the amyloid-beta (A beta) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimers disease (AD). A beta peptides are generated from proteolytic processing of the transmembrane A beta precursor protein (A beta PP) via sequential proteolysis through the beta-secretase activity of beta-site A beta PP-cleaving enzyme (BACE1) and by the intramembranous enzyme gamma-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 A beta peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human A beta PP is co-expressed with human BACE1, resulting in production of the A beta peptide through the processing of A beta PP by BACE1 and by endogenous fly gamma-secretase. Here, we performed a parallel study of flies that expressed the A beta(1-42) peptide alone or that co-expressed A beta PP and BACE1. Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the A beta(1-42), A beta(1-40) and A beta(1-38) peptides were examined. Our data reveal that the toxic effect per amount of detected A beta(1-42) peptide was higher in the flies co-expressing A beta PP and BACE1 than in the A beta(1-42)-expressing flies, and that the co-existence of A beta(1-42) and A beta(1-40) in the flies co-expressing A beta PP 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 A beta(1-42) 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.
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| 10. |
- Bergkvist, Liza, et al.
(författare)
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Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimers disease
- 2020
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Ingår i: FEBS Open Bio. - : WILEY. - 2211-5463. ; 10:3, s. 338-350
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimers 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 A beta fly model and the A beta PP-BACE1 fly model. In the A beta fly model, the A beta peptide is fused to a secretion sequence and directly overexpressed. In the A beta PP-BACE1 model, human A beta PP and human BACE1 are expressed in the fly, resulting in in vivo production of A beta peptides and other A beta PP 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 A beta fly model, this correlates with high A beta(1-42) levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the A beta PP-BACE1 fly model, neuronal cell death correlates with low A beta(1-42) levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of A beta PP/A beta antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the A beta PP-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.
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