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1.	Klionsky, Daniel J, et al. (författare) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). 2016 Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 12:1, s. 1-222 Tidskriftsartikel (refereegranskat)
2.	Klionsky, Daniel J., et al. (författare) Guidelines for the use and interpretation of assays for monitoring autophagy 2012 Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544 Forskningsöversikt (refereegranskat)abstract In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
3.	Agholme, Lotta, et al. (författare) Amyloid-β Secretion, Generation, and Lysosomal Sequestration in Response to Proteasome Inhibition : Involvement of Autophagy 2012 Ingår i: Journal of Alzheimer's Disease. - : I O S Press. - 1387-2877 .- 1875-8908. ; 31:2, s. 343-358 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.
4.	Agholme, Lotta, et al. (författare) An In Vitro Model for Neuroscience: Differentiation of SH-SY5Y Cells into Cells with Morphological and Biochemical Characteristics of Mature Neurons 2010 Ingår i: Journal of Alzheimer's Disease. - : Ios Press. - 1387-2877 .- 1875-8908. ; 20:4, s. 1069-1082 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.
5.	Agholme, Lotta, et al. (författare) Proteasome Inhibition Induces Stress Kinase Dependent Transport Deficits – Implications for Alzheimer’s Disease 2014 Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 58, s. 29-39 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.
6.	Agholme, Lotta (författare) The involvement of degradation pathways and neuron-to-neuron transmission in Alzheimer’s disease 2012 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Although the vast majority of Alzheimer’s disease (AD) cases are of the sporadic type, mutations causing the familial form have been the focus of AD research for decades. The disease is pathologically characterised by β-amyloid (Aβ) and tau protein aggregates in neuritic plaques and neurofibrillary tangles. Furthermore, it is known that AD pathology spreads throughout the brain, most often along the same anatomical pattern. However, so far no cause for the sporadic form of the disease has been found. Accumulation of protein aggregates as well as decreased activity of the protein degradation systems, lysosomes and proteasomes, is found in diseased brains. This indicates that defective degradation contributes to sporadic AD.The aim of this thesis was to develop an improved neuronal model, and study the effects of decreased proteasome function on tau phosphorylation and axonal transport. In addition, the effects on Aβ accumulation and generation upon proteasome inhibition were investigated. Finally, the possibility that intracellularly accumulated Aβ oligomers could be transferred from one neuron to another was tested.Differentiation of human SH-SY5Y neuroblastoma cells in an extracellular matrix gel, using a set of neurotrophic factors, resulted in cells with neuronal phenotype, expressing neuron specific markers and all six adult isoforms of tau. Within this neuronal model, we found that reduced proteasome activity inhibited neuritic transport, and caused tau phosphorylation in a c-Jun and ERK 1/2 dependent manner. Using proteasome inhibition in APP overexpressing cells, we found an autophagy dependent intralysosomal Aβ accumulation, together with elevation of intra- and extracellular concentrations of Aβ. Autophagy inhibition protected the cells from the toxicity induced by decreased proteasome activity. Finally, we could, as the first group, show that Aβ can be directly transferred from one neuron to another through connected neurites. Furthermore, accumulation of Aβ in the endo-lysosomal compartment of receiving cells caused toxicity and neurodegeneration.We believe that cells not able to degrade accumulated Aβ, due to increased generation or reduced degradative capacity, instead tries to clear its content through transfer to connected neurons. If not properly degraded in the receiving cell, this can accelerate AD pathology and cause neuritic and neuronal degeneration spreading throughout the brain. Increasing the activity of the degradative systems, or inhibiting transmission of Aβ between neurons could therefore be novel treatments for AD.
7.	Appelqvist, Hanna, et al. (författare) Attenuation of the Lysosomal Death Pathway by Lysosomal Cholesterol Accumulation 2011 Ingår i: American Journal of Pathology. - : American Society for Investigative Pathology (ASIP). - 0002-9440 .- 1525-2191. ; 178:2, s. 629-639 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.
8.	Appelqvist, Hanna, 1981- (författare) Lysosomal Membrane Stability and Cathepsins in Cell Death 2012 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Lysosomes are acidic organelles that are critically involved in a number of physiological processes, including macromolecule degradation, endocytosis, autophagy, exocytosis and cholesterol homeostasis. Several pathological conditions, such as cancer, neurodegenerative disorders and lysosomal storage diseases, involve lysosomal disturbances, indicating the importance of the organelle for correct cellular function. The aim of this thesis was to investigate the role of lysosomes in cell death signaling.Previous studies have shown that permeabilization of the lysosomal membrane and release of hydrolytic enzymes such as cathepsin D to the cytosol occurs during apoptosis. We identified Bid and 14-3-3 proteins as cytosolic targets of cathepsin D in human fibroblasts. Truncated Bid, generated by cathepsin D proteolytic cleavage, stimulates Bax-mediated release of pro-apoptotic factors from the mitochondria, thereby engaging the intrinsic pathway to apoptosis.Since the presence of cathepsins in the cytosol is sufficient to induce apoptosis, the permeability of the lysosomal membrane influences the fate of the cell. In this thesis, we demonstrated that the stability of the lysosomal membrane can be manipulated by altering the lysosomal cholesterol content. Cells with high lysosomal cholesterol content were less prone to undergo apoptosis when challenged with stimuli known to induce lysosome-mediated cell death. In addition, cholesterol accumulation was associated with increased expression of lysosome-associated membrane proteins and storage of other lipids; however, these factors did not contribute to lysosomal stabilization.Lysosomal membrane permeabilization and cathepsins contribute to ultraviolet (UV) irradiation-induced apoptosis. We demonstrate plasma membrane damage induced by UVA irradiation to be rapidly repaired by lysosomal exocytosis in human keratinocytes. Despite efficient plasma membrane resealing, the cells underwent apoptosis, which was dependent on early activation of caspase-8. The activation of caspase-8 was lysosome-dependent and occurred in vesicles positive for lysosomal markers.This thesis demonstrates the importance of lysosomal stability for apoptosis regulation and that this stability can be influenced by drug intervention. Modulation of the lysosomal membrane permeability may have potential for use as a therapeutic strategy in conditions associated with accelerated or repressed apoptosis.
9.	Appelqvist, Hanna, et al. (författare) Lysosome-Mediated Apoptosis is Associated with Cathepsin D-Specific Processing of Bid at Phe24,Trp48, and Phe183 2012 Ingår i: Annals of Clinical and Laboratory Science. - : Institute for Clinical Science. - 0091-7370 .- 1550-8080. ; 42:3, s. 231-242 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.
10.	Appelqvist, Hanna, et al. (författare) Sensitivity to Lysosome-Dependent Cell Death is Directly Regulated by Lysosomal Cholesterol Content 2012 Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:11 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.
11.	Appelqvist, Hanna, et al. (författare) The lysosome: from waste bag to potential therapeutic target 2013 Ingår i: Journal of Molecular Cell Biology. - : Oxford University Press (OUP): Policy B - Oxford Open Option D. - 1674-2788 .- 1759-4685. ; 5:4, s. 214-226 Forskningsöversikt (refereegranskat)abstract 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.
12.	Armstrong, Andrea, et al. (författare) Lysosomal Network Proteins as Potential Novel CSF Biomarkers for Alzheimers Disease 2014 Ingår i: Neuromolecular medicine. - : Humana Press. - 1535-1084 .- 1559-1174. ; 16:1, s. 150-160 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.
13.	Bergkvist, Liza, et al. (författare) 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 Ingår i: BIOLOGY OPEN. - : COMPANY OF BIOLOGISTS LTD. - 2046-6390. ; 5:8, s. 1030-1039 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.
14.	Bergkvist, Liza, 1985- (författare) Amyloid-β and lysozyme proteotoxicity in Drosophila : Beneficial effects of lysozyme and serum amyloid P component in models of Alzheimer’s disease and lysozyme amyloidosis 2017 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.
15.	Bergkvist, Liza, et al. (författare) Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimers disease 2020 Ingår i: FEBS Open Bio. - : WILEY. - 2211-5463. ; 10:3, s. 338-350 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.
16.	Bivik, Cecilia, et al. (författare) UVA/B induced apoptosis in human melanocytes involves translocation of cathepsins and Bcl-2 family members 2006 Ingår i: Journal of Investigative Dermatology. - : Elsevier BV. - 0022-202X. ; 126:5, s. 1119-1127 Tidskriftsartikel (refereegranskat)abstract We demonstrate UVA/B to induce apoptosis in human melanocytes through the mitochondrial pathway, displaying cytochrome c release, caspase-3 activation, and fragmentation of nuclei. The outcome of a death signal depends on the balance between positive and negative apoptotic regulators, such as members of the Bcl-2 protein family. Apoptotic melanocytes, containing fragmented nucleus, show translocation of the proapoptotic proteins Bax and Bid from the cytosol to punctate mitochondrial-like structures. Bcl-2, generally thought to be attached only to membranes, was in melanocytes localized in the cytosol as well. In the fraction of surviving melanocytes, that is, cells with morphologically unchanged nucleus, the antiapoptotic proteins Bcl-2 and Bcl-XL were translocated to mitochondria following UVA/B. The lysosomal proteases, cathepsin B and D, which may act as proapoptotic mediators, were released from lysosomes to the cytosol after UVA/B exposure. Proapoptotic action of the cytosolic cathepsins was confirmed by microinjection of cathepsin B, which induced nuclear fragmentation. Bax translocation and apoptosis were markedly reduced in melanocytes after pretreatment with either cysteine or aspartic cathepsin inhibitors. No initial caspase-8 activity was detected, excluding involvement of the death receptor pathway. Altogether, our results emphasize translocation of Bcl-2 family proteins to have central regulatory functions of UV-induced apoptosis in melanocytes and suggest cathepsins to be proapoptotic mediators operating upstream of Bax.
17.	Boman, Andrea, et al. (författare) Distinct lysosomal network protein profiles in parkinsonian syndrome cerebrospinal fluid 2016 Ingår i: Journal of Parkinson's Disease. - : IOS Press. - 1877-7171 .- 1877-718X. ; 6:2, s. 307-315 Tidskriftsartikel (refereegranskat)abstract Introduction: Clinical diagnosis of parkinsonian syndromes like Parkinson’s disease, corticobasal degeneration and progressive supranuclear palsy is hampered by overlapping symptomatology and lack of biomarkers for diagnosis, and definitive diagnosis is only possible post-mortem. Since impaired protein degradation plays an important role in many neurodegenerative disorders, we hypothesized that levels and profiles of lysosomal network proteins in cerebrospinal fluid could be changed in these parkinsonian syndromes.Methods: Cerebrospinal fluid samples were collected from Parkinson’s disease patients (n=18), clinically diagnosed 4-repeat tauopathy patients, corticobasal syndrome (n=6) and progressive supranuclear palsy (n=5), pathologically diagnosed progressive supranuclear palsy (n=8) and corticobasal degeneration patients (n=7). Each patient set was compared to its appropriate control group consisting of the same number of age and gender matched individuals. Lysosomal network protein levels were detected via Western blotting.Results: Lysosomal network proteins have markedly different cerebrospinal fluid protein levels and profiles in Parkinson’s disease, corticobasal degeneration and progressive supranuclear palsy. Lysosomal-associated membrane proteins 1 and 2 were significantly decreased in Parkinson´s disease; early endosomal antigen 1 was decreased and lysozyme increased in progressive supranuclear palsy; and lysosomal-associated membrane proteins 1 and 2, microtubule-associated protein 1 light chain 3 and lysozyme were increased in corticobasal degeneration.Conclusions: Lysosomal network proteins hold promise of being interesting novel candidates for biomarker studies and for elucidating disease mechanisms of Parkinson’s disease, corticobasal degeneration and progressive supranuclear palsy, but further validation studies will be needed to assess the specificity and the predictive value of these proteins in CSF.
18.	Boman, Andrea, 1978- (författare) Lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease 2015 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract The pre-symptomatic stage of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) occurs several decades before the clinical onset. Changes in the lysosomal network, i.e. the autophagosomal, endosomal and lysosomal vesicular system, are among the first alterations observed. There are currently no treatments to slow or cure neurodegenerative diseases, and there is a great need for discovery of treatment targets in cellular pathways where pathology pre-dates the neuronal death. It is also crucial to be able to diagnose neurodegenerative diseases earlier, both to enable early intervention treatment and aid in selecting clinical trial populations before the patient has widespread pathology.This thesis aims at investigating the potential of lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease.A targeted search for lysosomal network proteins was performed in cerebrospinal fluid (CSF) from AD patients, and seven proteins: early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), lysozyme, microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were elevated. The levels of EEA1, LAMP-1, LAMP-2, LC3, lysozyme and Rab3 were also measured in CSF from parkinsonian syndrome patients: PD, clinically diagnosed 4-repeat tauopathy, pathologically confirmed corticobasal degeneration (CBD) and pathologically confirmed progressive supranuclear palsy (PSP) patients. LAMP-1 and LAMP-2 were decreased in PD. LC3 and lysozyme levels were increased in 4-repeat tauopathy patients. EEA1 was decreased and lysozyme increased in PSP, and LAMP-1, LAMP-2, LC3 and lysozyme were increased in CBD. The lysosomal network proteins had different CSF protein profiles in all the parkinsonian syndromes, as well as in AD. It should be emphasized that only a select few of the lysosomal network proteins were observed to be changed, rather than a general change in lysosomal network proteins, which implicates the involvement of these seven proteins in specific pathological processes. The most interesting candidates, LAMP-2 and lysozyme, were selected for further study for their involvement in the pathology of AD.Lysozyme was found to co-localise with Aβ plaques in AD patients and overexpression prolonged survival and improved the activity in a Drosophila model of AD. Lysozyme was found to alter the aggregation pathway of Aβ1-42, to counteract the formation of toxic Aβ species and to protect from Aβ1-42 induced cell toxicity. Aβ1-42 in turn was found to increase the expression of lysozyme in both neuronal and glial cells. These data suggest that lysozyme levels rise in AD as a compensatory response which is protective against Aβ associated toxicity.LAMP-2 mRNA and protein were found increased in brain areas relevant for AD pathology and various cellular models showed complex involvement of LAMP-2 in Aβ related pathology, with extensive crosstalk between LAMP-2 and Aβ. Exposure to oligomeric Aβ1-42 caused an upregulation of LAMP-2 and in turn, overexpression of LAMP-2 caused a reduction in secreted levels of Aβ1-42, as well as changing the generation pattern of Aβ and affecting clearance and secretion of Aβ1-42. These data indicate that the increased levels of LAMP-2 in AD could be an attempt to regulate Aβ generation and secretion.In summary, this thesis reports that utilising lysosomal network proteins as biomarkers and novel therapeutic targets for neurodegenerative diseases holds great promise.
19.	Boman, Andrea, et al. (författare) The role of LAMP-2 in AβPP processing and Aβ degradation; implications for Alzheimer’s Disease 2015 Annan publikation (övrigt vetenskapligt/konstnärligt)abstract Dysfunction in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are implicated in the pathways in Alzheimer’s disease brain pathology. This dysfunction is mirrored in the cerebrospinal fluid where a specific subset of lysosomal network proteins are found at elevated levels, lysosomal associated membrane protein-2 (LAMP-2) being one of the identified lysosomal proteins. Here we report that hippocampus and frontal cortex in Alzheimer’s disease cases have increased mRNA and protein expression of LAMP-2, and thus these brain areas are likely involved in the increased LAMP-2 levels seen in cerebrospinal fluid from Alzheimer’s disease patients. The increased LAMP-2 levels correlated with increased levels of β-amyloid1-42 (Aβ1-42). Oligomeric Aβ1-42 caused an upregulation of intracellular LAMP-2 in neuroblastoma cells, but did not trigger the release of LAMP-2 to the extracellular milieu, indicating that other cell types or mechanisms are responsible for the LAMP-2 release seen in cerebrospinal fluid. Overexpression of LAMP-2 in neuroblastoma cells caused a trend of reduction of secreted Aβ1-42 and changed the processing pattern of the Aβ precursor protein. These results indicate that Aβ1-42 mediated increase of LAMP-2 expression can act as a regulator of Aβ generation and secretion. LAMP-2 overexpression did not change the cellular uptake of extracellularly added Aβ1-42, but caused a delayed clearance of Aβ1-42. Whether the prolonged intracellular localization of Aβ1-42 in LAMP-2 overexpressing cells can change the transmission or degradation of Aβ remains to be investigated.
20.	Civitelli, Livia, et al. (författare) The Luminescent Oligothiophene p-FTAA Converts Toxic A beta(1-42) Species into Nontoxic Amyloid Fibers with Altered Properties 2016 Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 291:17, s. 9233-9243 Tidskriftsartikel (refereegranskat)abstract Aggregation of the amyloid-(beta) peptide (A beta) 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 A beta 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 A beta fibrillation pathway may be a valid approach to reduce A beta 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 A beta species in favor of the formation of larger insoluble nontoxic amyloid fibrils, there by counteracting A beta-mediated cytotoxicity. Moreover, p-FTAA bound to early formed A beta species and induced a rapid formation of beta-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 A beta species that were nontoxic which indicates that p-FTAA might have therapeutic potential.
21.	Dilna, Aysha, et al. (författare) Amyloid-beta induced membrane damage instigates tunneling nanotube-like conduits by p21-activated kinase dependent actin remodulation 2021 Ingår i: Biochimica et Biophysica Acta - Molecular Basis of Disease. - : ELSEVIER. - 0925-4439 .- 1879-260X. ; 1867:12 Tidskriftsartikel (refereegranskat)abstract Alzheimers disease (AD) pathology progresses gradually via anatomically connected brain regions. Direct transfer of amyloid-beta(1-42) oligomers (oA beta) between connected neurons has been shown, however, the mechanism is not fully revealed. We observed formation of oA beta induced tunneling nanotubes (TNTs)-like nanoscaled f-actin containing membrane conduits, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that oA beta propagate from one cell to another via TNT-like structures. Preceding the formation of TNT-like conduits, we detected oA beta_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 oA beta 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 oA beta 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 oA beta 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 oA beta 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.
22.	Englund, Ulrika, et al. (författare) A Voltage Dependent Non-Inactivating Na+ Channel Activated during Apoptosis in Xenopus Oocytes 2014 Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 9:2, s. 0088381- Tidskriftsartikel (refereegranskat)abstract 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 mM) and amiloride (10 mM), while the Ca2+-channel blocker verapamil (50 mM) 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+ whith 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.
23.	Englund, Ulrika (författare) The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes 2014 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration.
24.	Fahlgren, Anna, 1972-, et al. (författare) Design and Outcome of a CDIO Syllabus Survey for a Biomedicine Program 2019 Ingår i: The 15th International CDIO Conference: Proceedings – Full Papers. - Aarhus : Aarhus University. - 9788775074594 ; , s. 191-200 Konferensbidrag (refereegranskat)abstract The CDIO Syllabus survey has successfully been applied to the Bachelor’s and Master’s programs in Experimental and Medical Biosciences, within the Faculty of Medicine and Health Sciences at Linköping University, Sweden. The programs are and have been, subject to considerable redesign with strong influence from the CDIO framework. One of the main drivers for the redesign is a shift concerning the main job market after graduation, from an academic career to industry and healthcare. One of the steps in the development process has been to carry out a Syllabus survey based on an adapted version of the CDIO Syllabus. The survey was sent out to students and to various categories of professionals, and in total 87 responses were received. The adapted version of the Syllabus and the design, execution, and outcome of the survey is presented.
25.	Göransson, Anna-Lena, et al. (författare) Identification of distinct physiochemical properties of the toxic prefibrillar species formed by Aβ peptide variants 2012 Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Wiley-Blackwell. - 0006-291X .- 1090-2104. ; 420:4, s. 895-900 Tidskriftsartikel (refereegranskat)abstract The formation of amyloid-β peptide (Aβ) 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 Aβ. 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 Aβ-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various Aβ 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 Aβ 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 Aβ peptide to form nontoxic versus toxic species.
26.	Helmfors, Linda, et al. (författare) A protective role of lysozyme in Alzheimer disease Annan publikation (övrigt vetenskapligt/konstnärligt)abstract Alzheimer disease (AD) is a devastating neurodegenerative disorder where extracellular plaques composed of amyloid β (Aβ) peptides and neuroinflammation are some of the main hallmarks of the disease. Activated microglial cells, which are the resident macrophages in the central nervous system, are suggested to trigger the inflammation response in AD. To discover neuroinflammation biomarkers would be important to reveal the pathological mechanisms of AD and develop therapies that target inflammation mediators. Lysozyme is part of the innate immune system and is secreted from macrophages during various inflammation conditions. However, the involvement of lysozyme in AD pathology has not been explored previously. We have discovered that lysozyme is up-regulated in cerebrospinal fluid from AD patients. Cells exposed to Aβ increased the expression of lysozyme indicating that Aβ might be responsible for the upregulation of lysozyme detected in cerebrospinal fluid. In vitro studies revealed that lysozyme binds to monomeric Aβ1-42 and alters the aggregation pathway counteracting formation of toxic Aβ species. In a newly developed Drosophila model, co-expression of lysozyme with Aβ in brain neurons reduced the formation of insoluble Aβ species, prolonged the survival and improved the activity of the double transgenic flies compared to flies only expressing Aβ. Our findings identify lysozyme as a modulator of Aβ aggregation and toxicity and our discoveries has the potential to be used for development of new treatment strategies and to use lysozyme as a biomarker for AD.
27.	Helmfors, Linda, et al. (författare) Protective properties of lysozyme on β-amyloid pathology : implications for Alzheimer disease 2015 Ingår i: Neurobiology of Disease. - : Elsevier. - 0969-9961 .- 1095-953X. ; 83, s. 122-133 Tidskriftsartikel (refereegranskat)abstract The hallmarks of Alzheimer disease are amyloid-β 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-β1-40 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-β in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-β1-42 reduced the formation of soluble and insoluble amyloid-β species, prolonged survival and improved the activity of amyloid-β1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-β increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-β1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-β species. Overall, these studies establish a protective role for lysozyme against amyloid-β 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.
28.	Johansson, Ann-Charlotte, et al. (författare) Cathepsin D-mediated processing of Bid at Phe24, Trp48, and Phe183 2008 Ingår i: International Journal of Experimental Pathology. Tidskriftsartikel (refereegranskat)
29.	Johansson, Ann-Charlotte, et al. (författare) Regulation of apoptosis-associated lysosomal membrane permeabilization 2010 Ingår i: APOPTOSIS. - : Springer Science Business Media. - 1360-8185 .- 1573-675X. ; 15:5, s. 527-540 Tidskriftsartikel (refereegranskat)abstract Lysosomal membrane permeabilization (LMP) occurs in response to a large variety of cell death stimuli causing release of cathepsins from the lysosomal lumen into the cytosol where they participate in apoptosis signaling. In some settings, apoptosis induction is dependent on an early release of cathepsins, while under other circumstances LMP occurs late in the cell death process and contributes to amplification of the death signal. The mechanism underlying LMP is still incompletely understood; however, a growing body of evidence suggests that LMP may be governed by several distinct mechanisms that are likely engaged in a death stimulus- and cell-type-dependent fashion. In this review, factors contributing to permeabilization of the lysosomal membrane including reactive oxygen species, lysosomal membrane lipid composition, proteases, p53, and Bcl-2 family proteins, are described. Potential mechanisms to safeguard lysosomal integrity and confer resistance to lysosome-dependent cell death are also discussed.
30.	Kim, Woojin Scott, et al. (författare) Alpha-synuclein biology in Lewy body diseases. 2014 Ingår i: Alzheimer's research & therapy. - : BioMed Central. - 1758-9193. ; 6:5 Tidskriftsartikel (refereegranskat)abstract 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.
31.	Kim, Woojin Scott, et al. (författare) Increased ATP-binding cassette transporter A1 expression in Alzheimer's disease hippocampal neurons 2010 Ingår i: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 21:1, s. 193-205 Tidskriftsartikel (refereegranskat)abstract ATP-binding cassette transporter A1 (ABCA1) reduces amyloid-beta burden in transgenic mouse models of Alzheimer's disease (AD). Associations between ABCA1 polymorphisms and AD risk are also established. Little is known regarding the regulation of ABCA1 expression in the brain and how this may be affected by AD. In the present study we assessed ABCA1 mRNA and protein expression in the hippocampus of AD cases compared to controls. ABCA1 was clearly expressed in hippocampal neurons and expression was increased two- to three-fold in AD cases. The increased hippocampal ABCA1 expression was associated with increased APOE and PUMA gene expression, implying an association with neuronal stress. Consistent with this, treatment of SK-N-SH neurons with amyloid-beta peptide resulted in a 48% loss in survival and a significant upregulation of ABCA1, APOE, and PUMA gene expression. Studies in young (2 month) and old (12 month) transgenic mice expressing a familial AD form of human amyloid-beta protein precursor and presenilin-1 revealed a significant age-dependent upregulation of hippocampal Abca1 compared to wild-type control mice. However, hippocampal Apoe and Puma gene expression were not correlated with increased Abca1 expression in mice. Our data indicate that ABCA1 is upregulated in AD hippocampal neurons potentially via an amyloid-beta-mediated pathway.
32.	Kågedal, Katarina, 1970-, et al. (författare) Anthraquinone toxicity and apoptosis in primary cultures of rat hepatocytes. 1999 Ingår i: Free radical research. - 1071-5762 .- 1029-2470. ; 31, s. 419-428 Tidskriftsartikel (refereegranskat)
33.	Kågedal, Katarina, 1970- (författare) Cathepsin D released from lysosomes mediates apoptosis 2003 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Last year (2002), the Nobel Prize in Physiology or Medicine was awarded to three scientists who have conducted pioneer research on programmed cell death. In the human body, more than a thousand billion cells are created every day, and an equal number die, thus programmed cell death, or apoptosis, is an important mechanism for maintaining tissue homeostasis and protecting against disease. Malfunctioning apoptosis is associated with many pathological conditions, for example, excess apoptosis is characteristic of AIDS, stroke, neurodegenerative diseases, and myocardinal infarction, and insufficient apoptosis is seen in autoimmune conditions and cancer. Robert Horvitz, one of the mentioned Nobel Prize Laureates, was the first to identify death genes, namely ced-3, -4, and -9 in the nematode Caenorhabditis elegans, which were later discovered to have counterparts in humans.The aim of this thesis is to clarify the participation of lysosomes and lysosomal proteases in the initiation of apoptosis. The lysosomal enzyme cathepsin D regulates the human homologue of ced-3, which encoded the caspase family of proteases. Moreover, the human homologue of ced-9 encodes the Bcl-2 family of proteins such as Bax, which was involved in regulating the release of cathepsin D from lysosomes during apoptosis. In the present studies, apoptosis was induced by various substances, all of which first caused damage to lysosomes with ensuing release of lysosomal proteases. Fibroblasts exposed either to free radicals generated by the redox cycling quinone naphthazarin or to the kinase inhibitor staurosporine exhibited rapid translocation of cathepsin D from lysosomes to the cytosol and subsequent apoptosis. Malignant macrophages (J774 cells) and T lymphocytes (Jurkat cells) exposed to the lysosomotropic detergent sphingosine displayed early lysosomal destabilization and later apoptosis. Sphingosine also destabilized isolated lysosomes. Moreover, mimicking the translocation of cathepsin D by microinjecting cathepsin D into the cytosol induced apoptosis in fibroblasts.In the mentioned systems, lysosomes were destabilized before mitochondrial changes occurred and caspases were activated. Furthermore, apoptosis was prevented by inhibition of cathepsin D in the naphthazarin, staurosporine, and sphingosine systems and by inhibition of cysteine proteases such as cathepsins B and L in the sphingosine system. These results emphasize that cytosolic localization of lysosomal proteases is necessary for the ability of these enzymes to induce apoptosis.The present results also demonstrate that, during apoptosis, lysosomal membranes are destabilized by the following: (i) free-radical-mediated lipid peroxidation; (ii) pore formation through the Bcl-2 family member Bax; (iii) the impact of the lysosomotropic detergent sphingosine. All three of these events have been implicated in numerous other apoptosis systems. Accordingly, the participation of lysosomal enzymes in apoptosis may be more widespread than previously assumed. This new perspective on lysosomes as regulators of apoptosis may lead to novel treatment strategies for diseases associated with malfunctioning apoptosis.
34.	Kågedal, Katarina, et al. (författare) Increased expression of the lysosomal cholesterol transporter NPC1 in Alzheimers disease 2010 Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : ELSEVIER SCIENCE BV. - 1388-1981 .- 1879-2618. ; 1801:8, s. 831-838 Tidskriftsartikel (refereegranskat)abstract The Niemann-Pick type Cl (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 Alzheimers 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.
35.	Kågedal, Katarina, et al. (författare) Lysosomal membrane permeabilization during apoptosis : Involvement of Bax? 2005 Ingår i: International journal of experimental pathology (Print). - : John Wiley & Sons. - 0959-9673 .- 1365-2613. ; 86:5, s. 309-321 Tidskriftsartikel (refereegranskat)abstract Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.
36.	Kågedal, Katarina, 1970-, et al. (författare) Sphingosine-induced apoptosis is dependent on lysosomal proteases 2001 Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 359:2, s. 335-343 Tidskriftsartikel (refereegranskat)abstract We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes.
37.	Kågedal, Katarina, et al. (författare) The lysosomal protease cathepsin D mediates apoptosis induced by oxidative stress 2001 Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 15:7, s. 1592-1594 Tidskriftsartikel (refereegranskat)
38.	Kågedal, Matts, et al. (författare) A positron emission tomography study in healthy volunteers to estimate mGluR5 receptor occupancy of AZD2066-Estimating occupancy in the absence of a reference region 2013 Ingår i: NeuroImage. - : Elsevier BV. - 1053-8119 .- 1095-9572. ; 82, s. 160-169 Tidskriftsartikel (refereegranskat)abstract AZD2066 is a new chemical entity pharmacologically characterized as a selective, negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). Antagonism of mGluR5 has been implicated in relation to various diseases such as anxiety, depression, and pain disorders. To support translation from preclinical results and previous experiences with this target in man, a positron emission tomography study was performed to estimate the relationship between AZD2066 plasma concentrations and receptor occupancy in the human brain, using the mGluR5 radioligand [C-11]-ABP688. The study involved PET scans on 4 occasions in 6 healthy volunteers. The radioligand was given as a tracer dose alone and following oral treatment with different doses of AZD2066. The analysis was based on the total volume of distribution derived fro m each PET-assessment. A non-linear mixed effects model was developed where ten delineated brain regions of interest from all PET scans were included in one simultaneous fit. For comparison the analysis was also performed according to a method described previously by Lassen et al. (1995). The results of the analysis showed that the total volume of distribution decreased with increasing drug concentrations in all regions with an estimated Kipl of 1170 nM. Variability between individuals and occasions in non-displaceable volume of distribution could explain most of the variability in the total volume of distribution. The Lassen approach provided a similar estimate for Kipl, but the variability was exaggerated and difficult to interpret.
39.	Kågedal, Matts, et al. (författare) Estimation of drug receptor occupancy when non-displaceable binding differs between brain regions : extending the simplified reference tissue model 2015 Ingår i: British Journal of Clinical Pharmacology. - : Wiley. - 0306-5251 .- 1365-2125. ; 80:1, s. 116-127 Tidskriftsartikel (refereegranskat)abstract AIM: The simplified reference tissue model (SRTM) is used for estimation of receptor occupancy assuming that the non-displaceable binding in the reference region is identical to the brain regions of interest. The aim of this work was to extended the SRTM to also account for inter-regional differences in non-displaceable concentrations, and to investigate if this model allowed estimation of receptor occupancy using white matter as reference. It was also investigated if an apparent higher affinity in caudate compared to other brain regions, could be better explained by a difference in the extent of non-displaceable binding.METHODS: The analysis was based on a PET study in 6 healthy volunteers using the 5-HT1B receptor radioligand [(11) C]AZ10419369. The radioligand was given intravenously as a tracer dose alone and following different oral doses of the 5-HT1B receptor antagonist AZD3783. Nonlinear mixed effects models were developed where differences between regions in non-specific concentrations were accounted for. The properties of the models were also evaluated by means of simulation studies.RESULTS: The estimate (95% CI) of KiPL was 10.2 ng/ml (5.4-15) and 10.4 ng/ml (8.1-13.6) based on the extended SRTM with white matter as reference and based on the SRTM using cerebellum as reference respectively. The estimate (95% CI) of KiPL for caudate relative to other brain regions was 55% ( 48% -62%).CONCLUSIONS: The extended SRTM allows consideration of white matter as reference region when no suitable grey matter region exists. The AZD3783 affinity appears to be higher in caudate compared with other brain regions.
40.	Kågedal, Matts, 1968-, et al. (författare) Extension of the simplified reference tissue model, to allow different non-specific concentrations in the brain region of interest and the reference region. Application to PET with the radioligand [11C]AZ10419369 displaced by AZD3783 at the serotonin 5HT1B receptor. Ingår i: NeuroImage. - 1053-8119 .- 1095-9572. Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
41.	Neuzil, Jiri, 1958-, et al. (författare) Vitamin E analogs : A new class of multiple action agents with anti-neoplastic and anti-atherogenic activity 2002 Ingår i: Apoptosis (London). - 1360-8185 .- 1573-675X. ; 7:2, s. 179-187 Tidskriftsartikel (refereegranskat)abstract The incidence of cancer and atherosclerosis, two most common causes of death in developed countries, has been stagnating or, even, increasing. Drugs effective against such conditions are needed and, in this regard, the potential anti-atherosclerotic activity of vitamin E analogs has been studied extensively. Surprisingly, recent results indicate that these agents may also exert anti-neoplastic effects. Here we review the evidence that particular analogs of vitamin E may act as both anti-atherogenic and anti-cancer agents, and discuss the possible molecular bases for these actions.
42.	Nilsson, Cathrine, 1978-, et al. (författare) Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry 2004 Ingår i: Methods in Cell Science. - : Springer Science and Business Media LLC. - 1381-5741 .- 1573-0603. ; 25:3-4, s. 185-194 Tidskriftsartikel (refereegranskat)abstract Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10ng/ml TNF- for 4h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2 ± 0.1 to 5.8s±0.1 and the lysosomal increased from 4.3±0.4 to 5.2±0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.
43.	Nilsson, Cathrine, 1978-, et al. (författare) Cytosolic acidification and lysosomal alkalinization during TNF-α induced apoptosis in U937 cells 2006 Ingår i: Apoptosis (London). - : Springer Netherlands. - 1360-8185 .- 1573-675X. ; 11:7, s. 1149-1159 Tidskriftsartikel (refereegranskat)abstract Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-α, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 ± 0.1 in healthy cells to 6.8 ± 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 ± 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F0F1-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 ± 0.3, in the healthy population, to 4.8 ± 0.3 and 5.5 ± 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-α.
44.	Nystrom, Sofie, et al. (författare) Monitoring amyloid formation and maturation in vitro and in vivo using LCO fluorescence in PRION, vol 6, issue , pp 13-13 2012 Ingår i: PRION. - : Landes Bioscience. ; , s. 13-13 Konferensbidrag (refereegranskat)abstract n/a
45.	Nyström, Sofie, et al. (författare) Properties of defined recombinant oligomeric forms of Aβ1‐42 Annan publikation (övrigt vetenskapligt/konstnärligt)abstract Oligomers of Aβ1-42 have been identified in human Alzheimer´s disease (AD) patients and in mouse models of AD. These species have attracted intense interest as possible neurological pathogens in AD. In our hands, expression of recombinant human Aβ1-42 in Escherichia coli followed by purification in the presence of cupric ions (CuCl2) afforded recovery of high quantities (>5 mg/L of culture) of well defined trimeric, hexameric, nonameric and dodecameric Aβ1-42. Strong denaturing conditions such as 6 M GuHCI, 8 M urea or boiling in 6.5 M urea supplemented with 2.5 % SDS all failed to separate the oligomers into smaller building blocks implicating that the oligomers are composed of covalently cross-linked Aβ1-42 monomers. Purification in the absence of cupric ions resulted in monomeric Aβ1-42. The Aβ1-42 oligomers were toxic and induced apoptosis when administered to neuroblastoma cells in culture. The described method producing oligomeric Aβ1-42 from a recombinant expression system paves the way for mechanistic studies, structural analysis, drug screening and opens up for vaccine development.
46.	Quinn, Carmel M., et al. (författare) Induction of fibroblast apolipoprotein E expression during apoptosis, starvation-induced growth arrest and mitosis 2004 Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 378:3, s. 753-761 Tidskriftsartikel (refereegranskat)abstract Apolipoprotein E (apoE) mediates the hepatic clearance of plasma lipoproteins, facilitates cholesterol efflux from macrophages and aids neuronal lipid transport. ApoE is expressed at high levels in hepatocytes, macrophages and astrocytes. In the present study, we identify nuclear and cytosolic pools of apoE in human fibroblasts. Fibroblast apoE mRNA and protein levels were up-regulated during staurosporine-induced apoptosis and this was correlated with increased caspase-3 activity and apoptotic morphological alterations. Because the transcription of apoE and specific pro-apoptotic genes is regulated by the nuclear receptor LXR (liver X receptor) α, we analysed LXRα mRNA expression by quantitative real-time PCR and found it to be increased before apoE mRNA induction. The expression of ABCA1 (ATP-binding cassette transporter A1) mRNA, which is also regulated by LXRα, was increased in parallel with apoE mRNA, indicating that LXRα probably promotes apoE and ABCA1 transcription during apoptosis. Fibroblast apoE levels were increased under conditions of serum-starvation-induced growth arrest and hyperoxia-induced senescence. In both cases, an increased nuclear apoE level was observed, particularly in cells that accumulated lipofuscin. Nuclear apoE was translocated to the cytosol when mitotic nuclear disassembly occurred and this was associated with an increase in total cellular apoE levels. ApoE amino acid sequence analysis indicated several potential sites for phosphorylation. In vivo studies, using 32P-labelling and immunoprecipitation, revealed that fibroblast apoE can be phosphorylated. These studies reveal novel associations and potential roles for apoE in fundamental cellular processes.
47.	Roberg, Karin, et al. (författare) Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblasts 2002 Ingår i: American Journal of Pathology. - 0002-9440 .- 1525-2191. ; 161:1, s. 89-96 Tidskriftsartikel (refereegranskat)abstract Recent reports have indicated that enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol early during apoptosis. We have previously noted that a translocation of cathepsins D and B occur before cytochrome c release and caspase activation in cardiomyocytes and human fibroblasts during oxidative stress-induced apoptosis. In the present report, we use a microinjection technique to investigate if cytosolic location of the cathepsins D and B are important for induction of apoptosis. We found that microinjection of cathepsin D into the cytosol of human fibroblasts caused apoptosis, which was detected as changes in distribution of cytochrome c, cell shrinkage, activation of caspases, chromatin condensation, and formation of pycnotic nuclei. No apoptosis was, however, induced by microinjection of cathepsin B. Moreover, apoptosis was prevented in fibroblasts pretreated with a caspase-3-like inhibitor, and also when microinjected with cathepsin D mixed with the cathepsin D inhibitor, pepstatin A. These results show that cytosolic cathepsin D can act as a proapoptotic mediator upstream of cytochrome c release and caspase activation in human fibroblasts.
48.	Sandin, Linnea, et al. (författare) Beneficial effects of increased lysozyme levels in Alzheimer’s disease modelled in Drosophila melanogaster 2016 Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 283:19, s. 3508-3522 Tidskriftsartikel (refereegranskat)abstract 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 Aβ1-42 or AβPP 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 Aβ1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1-42 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 Aβ1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1-42, which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.
49.	Sandin, Linnea, 1984- (författare) The influence of lysozyme and oligothiophenes on amyloid-β toxicity in models of Alzheimer’s disease 2016 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Alzheimer’s disease (AD) is a neurodegenerative disease and the most common cause of dementia worldwide. Apart from dominantly inherited mutations, age is the major risk factor and as life expectancy increases the prevalence for AD escalates dramatically. AD causes substantial problems for the affected persons and their families, and the society suffers economically. To date the available treatments only temporarily relieve the symptoms, wherefore the development of a cure is of utmost importance. The etiology of AD is still inconclusive but many believe that small aggregates (oligomers) of the protein amyloid-β (Aβ) are central for the onset of AD.The aims of this thesis were to investigate how different molecules affect the aggregation and toxicity of Aβ. In paper I and II, two oligothiophenes were studied; p-FTAA and h-FTAA and in paper III and IV the inflammatory protein lysozyme was explored. Differentiated neuroblastoma cells and Drosophila melanogaster were used as models of AD to address the issue.The results show that p-FTAA rescues neuroblastoma cells from Aβ toxicity when Aβ is coaggregated with lysozyme. Various biophysical studies show that the co-aggregation increases the formation of fibrillar Aβ structures rich in β-sheets. Noteworthy, these Aβ fibrils were more resistant to both degradation and denaturation, and less prone to propagate seeding from Aβ monomers. Furthermore, h-FTAA, but not p-FTAA, was able to protect neuroblastoma cell toxicity when exposed to Aβ with the Arctic mutation (AβArc), which probably reflects the weaker binding of AβArc to p-FTAA, compared to h-FTAA.Lysozyme levels were increased in CSF from patients that were both biochemically and clinically diagnosed with AD. In mice models of AD it was revealed that the mRNA increase in lysozyme correlates to increased Aβ pathology, but not to tau pathology, indicating that Aβ could drive the expression of lysozyme. To evaluate the effect for increased expression of lysozyme, co-expression of lysozyme was achieved in flies that expressed Aβ in the retina of the eyes, or in flies that expressed AβArc in the central nervous system. In all AD fly models, co-expression of lysozyme protected the cells from the Aβ induced toxicity. Of note, flies that expressed the toxic AβArc in the CNS of the flies showed an improvement in both lifespan and activity. Finally, we demonstrate that Aβ aggregating in the presence of lysozyme inhibits the cellular uptake of Aβ and also the cytotoxic effect of Aβ.The work included in this thesis demonstrates that the oligothiophenes p-FTAA and h-FTAA, and also lysozyme have the potential to be used as treatment strategies for sporadic AD, but remarkable, also in familial AD with the highly toxic Arctic mutation. The protective mechanism of p-FTAA seems to be attributed to the ability to generate stable Aβ fibrils with reduced seeding capacity, and that lysozyme inhibits the neuronal uptake of Aβ, which could prevent both the intracellular toxicity and cell-to-cell transmission of Aβ.
50.	Sandin, Linnea, et al. (författare) The Luminescent Conjugated Oligothiophene h-FTAA Attenuates the Toxicity of Different A beta Species 2021 Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 60:37, s. 2773-2780 Tidskriftsartikel (refereegranskat)abstract The prevailing opinion is that prefibrillar beta-amyloid (A beta) species, rather than end-stage amyloid fibrils, cause neuronal dysfunction in Alzheimers disease, although the mechanisms behind A beta 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 A beta(1- 42) and prion protein. In a previous study, we showed that an LCO, pentamer formyl thiophene acetic acid (p-FTAA), changed the toxicity of A beta(1-42). Here we investigated whether an LCO, heptamer formyl thiophene acetic acid (h-FTAA), could change the toxicity of A beta(1-42) by comparing its behavior with that of p-FTAA. Moreover, we investigated the effects on toxicity when A ss with the Arctic mutation (A beta Arc) was aggregated with both LCOs. Cell viability assays on SH-SY5Y neuroblastoma cells demonstrated that h-FTAA has a stronger impact on A beta(1-42) toxicity than does p-FTAA. Interestingly, h-FTAA, but not p-FTAA, rescued the A beta(Arc)-mediated toxicity. Aggregation kinetics and binding assay experiments with A beta(1-42) and A beta(Arc) 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 A beta(1-42) and A beta(Arc) toxicity, thus showing h-FTAA to be a useful tool for improving our understanding of the process of A beta aggregation linked to cytotoxicity.

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