| 11. |
- Nath, Sangeeta, et al.
(författare)
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Spreading of Neurodegenerative Pathology via Neuron-to-Neuron Transmission of beta-Amyloid
- 2012
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Ingår i: Journal of Neuroscience. - : SOC NEUROSCIENCE. - 0270-6474 .- 1529-2401. ; 32:26, s. 8767-8777
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimers disease (AD) is the major cause of dementia. During the development of AD, neurofibrillary tangles progress in a fixed pattern, starting in the transentorhinal cortex followed by the hippocampus and cortical areas. In contrast, the deposition of beta-amyloid (A beta) plaques, which are the other histological hallmark of AD, does not follow the same strict spatiotemporal pattern, and it correlates poorly with cognitive decline. Instead, soluble A beta oligomers have received increasing attention as probable inducers of pathogenesis. In this study, we use microinjections into electrophysiologically defined primary hippocampal rat neurons to demonstrate the direct neuron-to-neuron transfer of soluble oligomeric A beta. Additional studies conducted in a human donor-acceptor cell model show that this A beta transfer depends on direct cellular connections. As the transferred oligomers accumulate, acceptor cells gradually show beading of tubulin, a sign of neurite damage, and gradual endosomal leakage, a sign of cytotoxicity. These observations support that intracellular A beta oligomers play a role in neurodegeneration, and they explain the manner in which A beta can drive disease progression, even if the extracellular plaque load is poorly correlated with the degree of cognitive decline. Understanding this phenomenon sheds light on the pathophysiological mechanism of AD progression. Additional elucidation will help uncover the detailed mechanisms responsible for the manner in which AD progresses via anatomical connections and will facilitate the development of new strategies for stopping the progression of this incapacitating disease.
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| 12. |
- Ungerbäck, Jonas
(författare)
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Inflammation and Intestinal Homeostasis-Associated Genes in Colorectal Cancer
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is a global ‘killer’ and every year more than 1.2 million new individuals are affected and approximately 600 000 succumb to the disorder. Several mechanisms such as inactivation of tumor suppressor genes, activation of oncogenes and dysregulation of cell fate determinating pathways e.g. Wnt and Notch can initiate a cancerous cell growth and promote colorectal tumorigenesis. In addition, most tumors are exposed to an inflammatory environment, which together with the presence of mitogenic and angiogenic signals may sustain several hallmarks of cancer. Genetic alterations in inflammatory genes are associated with chronic inflammatory bowel disease, which is a strong risk factor of developing CRC. Scientists have for a long time looked for ‘the Key’ that would unlock the ‘cancer door’ but more likely cancer should be considered as not one but many diseases where almost every single patient is genetically and clinically unique. Hence recent research has turned to identify such inter-individual discrepancies and to find disease markers and strategies for guiding clinicians when tailoring individual management and optimized therapy. A deeper understanding of the regulation and genetic variation of inflammation and intestinal-homeostasis associated genes is pivotal to find potential targets for future therapies.The present thesis focuses on genetic variation and alterations in inflammatory genes as well as genes specifically involved in maintaining intestinal homeostasis. The most common anti-inflammatory drugs, NSAIDs, inhibit the prostanoid-generating COX-enzymes and are associated with decreased CRC risk when administered for a long time. Unfortunately, continuous NSAID treatment may lead to severe side-effects such as gastrointestinal bleeding, possibly through the ablation of non-PGE2 prostanoids. Therefore, a more specific inhibition of PGE2 has been suggested to be superior to classical NSAIDs. In papers I and II, the terminal PGE2 generating enzyme mPGES1 was studied in the context of intestinal cancer. Unexpectedly, ApcMin/+ mice with a targeted deletion of the mPGES1 encoding gene displayed significantly more and larger intestinal adenomas as compared to their wilde-type (wt) littermates. Probably this was due to the redirected generation of PGE2 towards non-PGE2 prostanoids seen in the murine tumors, resulting in enhanced pro-tumorigenic activity of these transmitter substances. Next, with a battery of functional and descriptive assays we investigated whether the outcome of mPGES1 expression and activity could depend on the genetic profile of the tumor e.g. the Apc mutational status. Indeed, high expression of mPGES1 was associated with the presence of wt-Apc, both in vitro and in vivo, most likely depending on mPGES1 mRNA stabilization rather than upregulation through β–catenin/Lef/Tcf4 signaling.NFκB is a major regulator of inflammation e.g. through the production of inflammatory cytokines. Variations in genes controlling inflammation and angiogenesis could potentially be used as biomarkers to identify patients with increased risk of CRC development, and/or to identify those with high risk of a rapidly progressing disease. Further, such analyzes have been suggested to select patients, which may benefit from specific anti-inflammatory or anti-angiogenic therapies. In paper III, genetic alterations in NFκB associated genes were studied among CRC patients and healthy controls. The NFκB negative regulator TNFAIP3 was found to exert tumor suppressive functions in CRC and moreover, homozygous mutant TNFAIP3 (rs6920220), homozygous mutant NFκB -94 ATTG ins/del and heterozygous NLRP3 (Q705K) were identified as prognostic markers for identifying CRC patients with a high risk of rapid progression. Further studies, which focus on the potential to treat such patients with anti-inflammatory IL-1β targeting therapies, are warranted.In the intestinal epithelium, Notch and Wnt signaling function in synergy to maintain homeostasis and together these pathways promote stem cell renewal and drive proliferation. Thus, dysregulation and/or overactivation of one of the two pathways could potentially lead to simultaneous activation of the other. While the genetic mechanisms explaining aberrant Wnt signaling in CRC are well-known, the reasons for the Notch pathway activation are less so. Further, relatively little is known about the mechanisms linking the two pathways in CRC. In paper IV, we addressed this question with a set of experimental in vitro assays, hereby identifying Notch2 together with several additional genes classically belonging to the Notch pathway, as putative targets for canonical and non-canonical Wnt signaling. We therefore suggest that aberrant Notch signaling in colon cancer cells may be the result of dysregulated Wnt signaling.In summary, the results here presented add a couple of pieces to the immensely complex jigsaw puzzle connecting intestinal homeostasis, inflammation and CRC. These results may aid in identifying future biomarkers or potential drug targets that could take us to the next level in the war against cancer.
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| 13. |
- Zheng, Lin, et al.
(författare)
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Intracellular distribution of amyloid beta peptide and its relationship to the lysosomal system.
- 2012
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Ingår i: Translational Neurodegeneration. - : BioMed Central. - 2047-9158. ; 1:1, s. 19-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundAmyloid beta peptide (Aβ) is the main component of extraneuronal senile plaques typical of Alzheimer’s disease (AD) brains. Although Aβ is produced by normal neurons, it is shown to accumulate in large amounts within neuronal lysosomes in AD. We have recently shown that under normal conditions the majority of Aβ is localized extralysosomally, while oxidative stress significantly increases intralysosomal Aβ content through activation of macroautophagy. It is also suggested that impaired Aβ secretion and resulting intraneuronal increase of Aβ can contribute to AD pathology. However, it is not clear how Aβ is distributed inside normal neurons, and how this distribution is effected when Aβ secretion is inhibited.MethodsUsing retinoic acid differentiated neuroblastoma cells and neonatal rat cortical neurons, we studied intracellular distribution of Aβ by double immunofluorescence microscopy for Aβ40 or Aβ42 and different organelle markers. In addition, we analysed the effect of tetanus toxin-induced exocytosis inhibition on the intracellular distribution of Aβ.ResultsUnder normal conditions, Aβ was found in the small cytoplasmic granules in both neurites and perikarya. Only minor portion of Aβ was colocalized with trans-Golgi network, Golgi-derived vesicles, early and late endosomes, lysosomes, and synaptic vesicles, while the majority of Aβ granules were not colocalized with any of these structures. Furthermore, treatment of cells with tetanus toxin significantly increased the amount of intracellular Aβ in both perikarya and neurites. Finally, we found that tetanus toxin increased the levels of intralysosomal Aβ although the majority of Aβ still remained extralysosomally.ConclusionOur results indicate that most Aβ is not localized to Golgi-related structures, endosomes, lysosomes secretory vesicles or other organelles, while the suppression of Aβ secretion increases intracellular intra- and extralysosomal Aβ.
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| 14. |
- Zheng, Lin, et al.
(författare)
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Intracellular localization of amyloid beta peptide in SH-SY5Y neuroblastoma cells
- 2013
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Ingår i: Journal of Alzheimer's Disease. - : IOS Press. - 1387-2877 .- 1875-8908. ; 37:4, s. 713-733
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid-beta peptide (A beta), the main component of Alzheimer's disease (AD) senile plaques, has been found to accumulate within the lysosomal compartment of AD neurons. We have previously shown that in differentiated SH-SY5Y neuroblastoma cells cultured under normal conditions, the majority of A beta is localized extralysosomally, while oxidative stress significantly increases intralysosomal A beta content through activation of macroautophagy. It is, however, not clear which cellular compartments contain extralysosomal A beta in intact SH-SY5Y cells, and how oxidative stress influences the distribution of extralysosomal A beta. Using confocal laser scanning microscopy and immunoelectron microscopy, we showed that in differentiated neuroblastoma cells cultured under normal conditions A beta (A beta(40), A beta(42), and A beta oligomers) is colocalized with both membrane-bound organelles (endoplasmic reticulum, Golgi complexes, multivesicular bodies/late endosomes, lysosomes, exocytotic vesicles and mitochondria) and non-membrane-bound cytosolic structures. Neuroblastoma cells stably transfected with A beta PP Swedish KM670/671NL double mutation showed enlarged amount of A beta colocalized with membrane compartments. Suppression of exocytosis by 5 nM tetanus toxin resulted in a significant increase of the amount of cytosolic A beta as well as A beta colocalized with exocytotic vesicles, endoplasmic reticulum, Golgi complexes, and lysosomes. Hyperoxia increased A beta localization in the endoplasmic reticulum, Golgi apparatus, mitochondria, and lysosomes, but not in the secretory vesicles. These results indicate that in SH-SY5Y neuroblastoma cells intracellular A beta is not preferentially localized to any particular organelle and, to a large extent, is secreted from the cells. Challenging cells to hyperoxia, exocytosis inhibition, or A beta overproduction increased intracellular A beta levels but did not dramatically changed its localization pattern.
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| 15. |
- Zheng, Lin
(författare)
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Lysosomal Involvement in the Pathogenesis of Alzheimer's Disease
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD), the major cause of senile dementia, is associated with progressive formation of neurofibrillary tangles and extraneuronal plaques composed of amyloid beta peptide (Aβ). Aβ has been also found within Alzheimer neurons in association with the lysosomal system, an acidic vacuolar compartment possessing numerous hydrolytic enzymes. Lysosomes have been shown to be involved in both the formation of Aβ and its toxicity to neurons. Another line of evidence implicates oxidative stress as an important factor in the development of AD. It is reported that oxidative damage is one of the earliest changes in AD and plays an important role in the development of the disease. Although both the lysosomal system and reactive oxygen species are involved in AD, the mechanisms of this involvement are not well understood.To gain insight into the relationship between oxidative stress and the lysosomal system in AD pathogenesis, we focused our study on: 1) The effect of oxidative stress on intracellular distribution of Aβ; 2) the role of endogenous Aβ in oxidant-induced apoptosis; 3) the role of autophagy and APP processing in oxidant induced damage; and, 4) the intraneuronal localization of Aβ and its relationship to the lysosomal system.In our study, hyperoxia (40% versus 8% ambient oxygen) was used as a model of mild oxidative stress in vitro, while transfected cells producing different amounts of Aβ were used to assess toxicity due to endogenous Aβ. It was found that: 1) oxidative stress induces autophagic uptake of Aβ, resulting in its partial accumulation within lysosomes; 2) oxidative stress can induce neuronal death through macroautophagy of Aβ and consequent lysosomal membrane permeabilization; 3) increased cellular Aβ production is associated with enhanced oxidative stress and enhanced macroautophagy, resulting in increased intralysosomal Aβ accumulation and consequent apoptosis; and, 4) in normal conditions, intracellular Aβ shows primarily cytosolic distribution, not related to lysosomes and other acidic vacuoles, endoplasmic reticulum, Golgi complexes, synaptic vesicles or mitochondria. Only a minor portion of Aβ shows partial colocalization with cellular organelles. Inhibition of secretion significantly increased Aβ colocalization with endoplasmic reticulum, Golgi complexes, synaptic vesicles and lysosomes, as well as the amount of mitochondrial and cytosolic Aβ.Oxidative stress induces intralysosomal autophagy-generated Aβ accumulation, consequently causing lysosomal membrane permeabilization and apoptosis. Our findings provide a possible explanation of the interactive role of oxidative stress and lysosomal system in AD pathogenesis, and may be helpful for a future therapeutic strategy against AD.
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| 16. |
- Zheng, Lin, et al.
(författare)
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Macroautophagy-generated increase of lysosomal amyloid beta-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells
- 2011
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 7:12, s. 1528-1545
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Tidskriftsartikel (refereegranskat)abstract
- Increasing evidence suggests the toxicity of intracellular amyloid beta-protein (A beta) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 400/c oxygen for five days, and consequent activation of macroautophagy and accumulation of A beta within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as A beta monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular A beta production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of v-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal A beta resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal A beta accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.
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| 17. |
- Zheng, Lin, et al.
(författare)
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Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells
- 2011
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Ingår i: Autophagy. - : Landes Bioscience. - 1554-8627 .- 1554-8635. ; 7:12, s. 1528-1545
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Tidskriftsartikel (refereegranskat)abstract
- Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days, and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide aditional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.
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