| 1. |
- Agholme, Lotta, et al.
(författare)
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An In Vitro Model for Neuroscience: Differentiation of SH-SY5Y Cells into Cells with Morphological and Biochemical Characteristics of Mature Neurons
- 2010
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Ingår i: Journal of Alzheimer's Disease. - : Ios Press. - 1387-2877 .- 1875-8908. ; 20:4, s. 1069-1082
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Tidskriftsartikel (refereegranskat)abstract
- Neuroscience, including research on Alzheimers disease, is hampered by the lack of suitable in vitro models to study the human nervous system. To counteract this, many attempts to differentiate cell lines into more neuron-like cells have been performed, resulting in partial expression of neuronal features. Furthermore, it has been reported that neuroblastoma cell lines lack mature isoforms of tau. Our aim was to develop an improved in vitro model, generating sustainable cells with morphology and biochemistry of human, mature neurons. To obtain cells with neuronal differentiation and function, we investigated the effect of combining three-dimensional culturing of SH-SY5Y cells in extracellular matrix (ECM) gel with several factors reported to have neuro-differentiating effects. This resulted in cells with apparent neuronal morphology with long, extensively branched neurites. Further investigation revealed expression of several neurospecific markers including synapse protein Sv2 and nuclear marker NeuN, as well as the presence of synapses and axonal vesicle transport. In addition, these cells expressed mature tau isoforms, and tau protein expression was significantly increased compared to undifferentiated cells, reaching levels found in adult human brain. In conclusion, we found that pre-treatment with retinoic acid followed by ECM gel culturing in combination with brain derived neurotrophic factor, neuregulin beta(1), nerve growth factor, and vitamin D-3 treatment generated sustainable cells with unambiguous resemblance to adult neurons. These cells also expresses adult splicing forms of tau with neuronal localization, making this cellular in vitro model useful in many areas of neuroscience research, particularly the Alzheimers disease field.
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| 2. |
- Agholme, Lotta, et al.
(författare)
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Amyloid-β Secretion, Generation, and Lysosomal Sequestration in Response to Proteasome Inhibition : Involvement of Autophagy
- 2012
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Ingår i: Journal of Alzheimer's Disease. - : I O S Press. - 1387-2877 .- 1875-8908. ; 31:2, s. 343-358
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Tidskriftsartikel (refereegranskat)abstract
- The proteasome is important for degradation of worn out and misfolded proteins. Decreased proteasome activity has been implicated in Alzheimer's disease (AD). Proteasome inhibition induces autophagy, but it is still unknown whether autophagy is beneficial or deleterious to AD neurons, as the autophagosome has been suggested as a site of amyloid-β (Aβ) generation. In this study, we investigated the effect of proteasome inhibition on Aβ accumulation and secretion, as well as the processing of amyloid-β protein precursor (AβPP) in AβPPSwe transfected SH-SY5Y neuroblastoma cells. We show that proteasome inhibition resulted in autophagy-dependent accumulation of Aβ in lysosomes, and increased levels of intracellular and secreted Aβ. The enhanced levels of Aβ could not be explained by increased amounts of AβPP. Instead, reduced degradation of the C-terminal fragment of AβPP (C99) by the proteasome makes C99 available for γ-secretase cleavage, leading to Aβ generation. Inhibition of autophagy after proteasome inhibition led to reduced levels of intracellular, but not secreted Aβ, and tended to further increase the C99 to AβPP ratio, supporting involvement of the autophagosome in Aβ generation. Furthermore, proteasome inhibition caused a reduction in cellular viability, which was reverted by inhibition of autophagy. Dysfunction of the proteasome could cause lysosomal accumulation of Aβ, as well as increased generation and secretion of Aβ, which is partly facilitated by autophagy. As a decrease in cellular viability was also detected, it is possible that upregulation of autophagy is an unsuccessful rescue mechanism, which instead of being protective, contributes to AD pathogenesis.
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| 3. |
- Agholme, Lotta, et al.
(författare)
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Proteasome Inhibition Induces Stress Kinase Dependent Transport Deficits – Implications for Alzheimer’s Disease
- 2014
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Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 58, s. 29-39
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, β-amyloid and hyperphosphorylated tau. Both cellular systems responsible for clearance of misfolded and aggregated proteins, the lysosomal and the proteasomal, have been shown to be malfunctioning in the aged brain and more so in AD patients. This malfunction could be the cause of β-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated how decreased proteasome activity affects AD related pathophysiological changes of microtubule transport and stability, as well as tau phosphorylation. To do this, we used our recently developed neuronal model where human SH-SY5Y cells obtain neuronal morphology and function through differentiation. We found that exposure to low doses of the proteasome inhibitor MG-115 caused disturbed neuritic transport, together with microtubule destabilization and tau phosphorylation. Furthermore, reduced proteasome activity activated several kinases implicated in AD pathology, including JNK, c-Jun and ERK 1/2. Restoration of the microtubule transport was achieved by inhibiting ERK 1/2 activation, and simultaneous inhibition of both ERK 1/2 and c-Jun reversed the proteasome inhibition-induced tau phosphorylation. Taken together, this study suggests that a decrease in proteasome activity can, through activation of c-Jun and ERK 1/2, result in several events contributing to AD pathology. Restoring proteasome function or inhibiting ERK 1/2 and c-Jun could therefore be used as novel treatments against AD.
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| 4. |
- Agholme, Lotta
(författare)
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The involvement of degradation pathways and neuron-to-neuron transmission in Alzheimer’s disease
- 2012
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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.
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| 5. |
- Domert, Jakob, et al.
(författare)
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Spreading of Amyloid-β Peptides via Neuritic Cell-to-cell Transfer Is Dependent on Insufficient Cellular Clearance
- 2014
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Ingår i: Neurobiology of Disease. - : Elsevier. - 0969-9961 .- 1095-953X. ; 65, s. 82-92
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Tidskriftsartikel (refereegranskat)abstract
- The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1-42 (oAβ1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1-42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.
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| 6. |
- 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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