scholarly journals Direct imaging of APP proteolysis in living cells

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3086 ◽  
Author(s):  
Niccoló Parenti ◽  
Ambra Del Grosso ◽  
Claudia Antoni ◽  
Marco Cecchini ◽  
Renato Corradetti ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Proteolytic Processing ◽  
Fluorescence Intensity Ratio ◽  
Green Fluorescence ◽  
Extracellular Medium ◽  
Human Neuroblastoma

Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α- and the β-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α-secretase cleavage without perturbing the β-secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

scholarly journals Direct imaging of APP proteolysis in living cells

2017 ◽  
Author(s):  
Niccoló Parenti ◽  
Ambra Del Grosso ◽  
Claudia Antoni ◽  
Marco Cecchini ◽  
Renato Corradetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Critical Issue ◽  
Living Cells ◽  
Proteolytic Processing ◽  
Fluorescence Intensity Ratio ◽  
Aβ Peptide ◽  
Green Fluorescence ◽  
Extracellular Medium ◽  
Human Neuroblastoma

It is now widely accepted that oligomers consisting of Aβ peptide are the cytotoxic species contributing mostly to the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal, respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1 fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

scholarly journals Direct imaging of APP proteolysis in living cells

2017 ◽  
Author(s):  
Niccoló Parenti ◽  
Ambra Del Grosso ◽  
Claudia Antoni ◽  
Marco Cecchini ◽  
Renato Corradetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Critical Issue ◽  
Living Cells ◽  
Proteolytic Processing ◽  
Fluorescence Intensity Ratio ◽  
Aβ Peptide ◽  
Green Fluorescence ◽  
Extracellular Medium ◽  
Human Neuroblastoma

It is now widely accepted that oligomers consisting of Aβ peptide are the cytotoxic species contributing mostly to the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal, respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1 fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

scholarly journals The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

1998 ◽  
Vol 9 (6) ◽  
pp. 1495-1512 ◽  
Author(s):  
Susanne Illenberger ◽  
Qingyi Zheng-Fischhöfer ◽  
Ute Preuss ◽  
Karsten Stamer ◽  
Karlheinz Baumann ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Cell Cycle ◽  
Alzheimer's Disease ◽  
Tau Protein ◽  
Chinese Hamster Ovary Cells ◽  
Phosphorylation Site ◽  
Chinese Hamster ◽  
Microtubule Assembly ◽  
Human Neuroblastoma

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

scholarly journals An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer's disease

2020 ◽  
Vol 295 (15) ◽  
pp. 4870-4880 ◽  
Author(s):  
Kazuma Murakami ◽  
Yayoi Obata ◽  
Asa Sekikawa ◽  
Haruka Ueda ◽  
Naotaka Izuo ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Senile Plaques ◽  
Neuroblastoma Cells ◽  
Physicochemical Characteristics ◽  
Rna Aptamers ◽  
Human Neuroblastoma ◽  
Conformationally Constrained ◽  
Preferential Binding

Oligomers of β-amyloid 42 (Aβ42), rather than fibrils, drive the pathogenesis of Alzheimer's disease (AD). In particular, toxic oligomeric species called protofibrils (PFs) have attracted significant attention. Herein, we report RNA aptamers with higher affinity toward PFs derived from a toxic Aβ42 dimer than toward fibrils produced from WT Aβ42 or from a toxic, conformationally constrained Aβ42 variant, E22P–Aβ42. We obtained these RNA aptamers by using the preincubated dimer model of E22P–Aβ42, which dimerized via a linker located at Val-40, as the target of in vitro selection. This dimer formed PFs during incubation. Several physicochemical characteristics of an identified aptamer, E22P–AbD43, suggested that preferential affinity of this aptamer toward PFs is due to its higher affinity for the toxic dimer unit (KD = 20 ± 6.0 nm) of Aβ42 than for less-toxic Aβ40 aggregates. Comparison of CD data from the full-length and random regions of E22P–AbD43 suggested that the preferential binding of E22P–AbD43 toward the dimer might be related to the formation of a G-quadruplex structure. E22P–AbD43 significantly inhibited the nucleation phase of the dimer and its associated neurotoxicity in SH-SY5Y human neuroblastoma cells. Of note, E22P–AbD43 also significantly protected against the neurotoxicity of WT Aβ42 and E22P–Aβ42. Furthermore, in an AD mouse model, E22P–AbD43 preferentially recognized diffuse aggregates, which likely originated from PFs or higher-order oligomers with curvilinear structures, compared with senile plaques formed from fibrils. We conclude that the E22P–AbD43 aptamer is a promising research and diagnostic tool for further studies of AD etiology.

scholarly journals Centella asiaticaExtract Improves Behavioral Deficits in a Mouse Model of Alzheimer's Disease: Investigation of a Possible Mechanism of Action

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Amala Soumyanath ◽  
Yong-Ping Zhong ◽  
Edward Henson ◽  
Teri Wadsworth ◽  
James Bishop ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Water Extract ◽  
Glutamate Toxicity ◽  
Tg2576 Mouse ◽  
Asiatic Acid ◽  
Human Neuroblastoma ◽  
Model Of Alzheimer’S Disease ◽  
Β Amyloid

Centella asiatica(CA), commonly named gotu kola, is an Ayurvedic herb used to enhance memory and nerve function. To investigate the potential use of CA in Alzheimer's disease (AD), we examined the effects of a water extract of CA (GKW) in the Tg2576 mouse, a murine model of AD with high β-amyloid burden. Orally administered GKW attenuated β-amyloid-associated behavioral abnormalities in these mice.In vitro, GKW protected SH-SY5Y cells and MC65 human neuroblastoma cells from toxicity induced by exogenously added and endogenously generated β-amyloid, respectively. GKW prevented intracellular β-amyloid aggregate formation in MC65 cells. GKW did not show anticholinesterase activity or protect neurons from oxidative damage and glutamate toxicity, mechanisms of current AD therapies. GKW is rich in phenolic compounds and does not contain asiatic acid, a known CA neuroprotective triterpene. CA thus offers a unique therapeutic mechanism and novel active compounds of potential relevance to the treatment of AD.

Cholinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In vitro Model for Alzheimer’s Disease Studies

2019 ◽  
Vol 56 (11) ◽  
pp. 7355-7367 ◽  
Author(s):  
Liana M. de Medeiros ◽  
Marco A. De Bastiani ◽  
Eduardo P. Rico ◽  
Patrícia Schonhofen ◽  
Bianca Pfaffenseller ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Line ◽  
In Vitro Model ◽  
Human Neuroblastoma ◽  
Vitro Model ◽  
Potential Use

scholarly journals Altered succinylation of mitochondrial proteins, APP and tau in Alzheimer’s disease

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Yun Yang ◽  
Victor Tapias ◽  
Diana Acosta ◽  
Hui Xu ◽  
Huanlian Chen ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Protein Modification ◽  
Proteolytic Processing ◽  
Microtubule Assembly ◽  
Mitochondrial Proteins ◽  
Protein Deposits ◽  
Abnormal Metabolism ◽  
Potential Link

AbstractAbnormalities in brain glucose metabolism and accumulation of abnormal protein deposits called plaques and tangles are neuropathological hallmarks of Alzheimer’s disease (AD), but their relationship to disease pathogenesis and to each other remains unclear. Here we show that succinylation, a metabolism-associated post-translational protein modification (PTM), provides a potential link between abnormal metabolism and AD pathology. We quantified the lysine succinylomes and proteomes from brains of individuals with AD, and healthy controls. In AD, succinylation of multiple mitochondrial proteins declined, and succinylation of small number of cytosolic proteins increased. The largest increases occurred at critical sites of amyloid precursor protein (APP) and microtubule-associated tau. We show that in vitro, succinylation of APP disrupted its normal proteolytic processing thereby promoting Aβ accumulation and plaque formation and that succinylation of tau promoted its aggregation to tangles and impaired microtubule assembly. In transgenic mouse models of AD, elevated succinylation associated with soluble and insoluble APP derivatives and tau. These findings indicate that a metabolism-linked PTM may be associated with AD.

Aromatase/Seladin-1 Interactions in Human Neuronal Cell Culture, the Hippocampus of Healthy Rats and Transgenic Alzheimer’s Disease Mice

Pharmacology ◽  
2018 ◽  
Vol 102 (1-2) ◽  
pp. 42-52 ◽  
Author(s):  
Hande Karahan ◽  
Sevda Lüle ◽  
Pelin Kelicen-Uğur
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Molecular Mechanisms ◽  
Neuronal Cell ◽  
Human Neuroblastoma ◽  
Protein Levels ◽  
Genes Encoding ◽  
Estrogen Biosynthesis

Background/Aims: Decreasing levels of aromatase and seladin-1 could be one of the molecular mechanisms of Alzheimer’s disease (AD). Aromatase is an enzyme that catalyzes estrogen biosynthesis from androgen precursors, and seladin-1 is an enzyme that converts desmosterol to cholesterol, which is the precursor of all hormones. Verifying the potential relationship between these proteins and accordingly determining new therapeutic targets constitute the aims of this study. Methods: Changes in protein levels were compared in vitro in aromatase and seladin-1 inhibitor-administered human neuroblastoma (SH-SY5Y) cells in vivo in intracerebroventricular (icv) aromatase or seladin-1 inhibitor-administered rats, as well as in transgenic AD mice in which the genes encoding these proteins were knocked out. Results and Conclusions: In the cell cultures, we observed that seladin-1 protein levels increased after aromatase enzyme inhibition. The hippocampal aromatase protein levels decreased following chronic seladin-1 inhibition in icv inhibitor-administered rats; however, the aromatase levels in the dentate gyrus of seladin-1 knockout (SelKO) AD male mice increased. These findings indicate a partial relationship between these proteins and their roles in AD pathology.

Proteolytic processing of the amyloid-beta protein precursor of Alzheimer's disease

2002 ◽  
Vol 38 ◽  
pp. 37-49 ◽  
Author(s):  
Janelle Nunan ◽  
David H Small
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Amyloid Beta ◽  
Alternative Pathway ◽  
Protein A ◽  
Proteolytic Processing ◽  
Gamma Secretase ◽  
Amyloid Beta Protein ◽  
Protein Precursor ◽  
Amyloid Beta Protein Precursor

The proteolytic processing of the amyloid-beta protein precursor plays a key role in the development of Alzheimer's disease. Cleavage of the amyloid-beta protein precursor may occur via two pathways, both of which involve the action of proteases called secretases. One pathway, involving beta- and gamma-secretase, liberates amyloid-beta protein, a protein associated with the neurodegeneration seen in Alzheimer's disease. The alternative pathway, involving alpha-secretase, precludes amyloid-beta protein formation. In this review, we describe the progress that has been made in identifying the secretases and their potential as therapeutic targets in the treatment or prevention of Alzheimer's disease.

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