Pharmacological inhibition of APP processing and knock-down of APP in primary human macrophages impairs the secretion of cytokines

Abstract Background The amyloid precursor protein (APP) and amyloid β (Aβ) peptides support the innate immune defense as an immune receptor, an antimicrobial peptide and an opsonine. In APP-deficient mouse models, a reduced secretion of cytokines has been observed. It is unclear whether this can be attributed to the lack of APP or to the missing secretion of Aβ peptides.Methods In primary human monocyte derived macrophages the secretion of Aβ peptides was inhibited by the γ-secretase inhibitor DAPT or by the β-secretase inhibitor GL-189, applied as a tripartite substance. Alternatively, APP was knocked down by transfection with siRNA. TNFα, IL-6 and IL-10 were measured by ELISA and the phagocytotic activity was evaluated by flow cytometry.Results Reduced concentrations of TNFα and IL-6 were observed in the media of APPk/d macrophages and after inhibition of the β-, or γ-secretase, especially after additional immunological activation with LPS. Secretion of IL-10 was increased after pharmacological inhibition of APP processing when the macrophages were not immunologically activated but was decreased during LPS-induced inflammation in APPk/d macrophages. No changes of the phagocytotic activity were observed.Conclusion We conclude that macrophage APP and Aβ peptides support the initiation of an immune response and are involved in the secretion of TNFα, IL-6 and IL-10.

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Enhancing α-secretase Processing for Alzheimer’s Disease—A View on SFRP1

Brain Sciences ◽

10.3390/brainsci10020122 ◽

2020 ◽

Vol 10 (2) ◽

pp. 122 ◽

Cited By ~ 1

Author(s):

Bor Luen Tang

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Amyloid Β ◽

Experimental Therapeutics ◽

Related Protein ◽

Endogenous Inhibitor ◽

Aβ Peptides ◽

App Processing ◽

Promising Therapeutic Target ◽

A Disintegrin And Metalloproteinase

Amyloid β (Aβ) peptides generated via sequential β- and γ-secretase processing of the amyloid precursor protein (APP) are major etiopathological agents of Alzheimer’s disease (AD). However, an initial APP cleavage by an α-secretase, such as the a disintegrin and metalloproteinase domain-containing protein ADAM10, precludes β-secretase cleavage and leads to APP processing that does not produce Aβ. The latter appears to underlie the disease symptom-attenuating effects of a multitude of experimental therapeutics in AD animal models. Recent work has indicated that an endogenous inhibitor of ADAM10, secreted-frizzled-related protein 1 (SFRP1), is elevated in human AD brains and associated with amyloid plaques in mouse AD models. Importantly, genetic or functional attenuation of SFRP1 lowered Aβ accumulation and improved AD-related histopathological and neurological traits. Given SFRP1′s well-known activity in attenuating Wnt signaling, which is also commonly impaired in AD, SFRP1 appears to be a promising therapeutic target for AD. This idea, however, needs to be addressed with care because of cancer enhancement potentials resulting from a systemic loss of SFRP1 activity, as well as an upregulation of ADAM10 activity. In this focused review, I shall discuss α-secretase-effected APP processing in AD with a focus on SFRP1, and explore the contrasting perspectives arising from the recent findings.

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The cellular expression and proteolytic processing of the amyloid precursor protein is independent of TDP-43

Bioscience Reports ◽

10.1042/bsr20200435 ◽

2020 ◽

Vol 40 (4) ◽

Cited By ~ 1

Author(s):

David A. Hicks ◽

Alys C. Jones ◽

Stuart M. Pickering-Brown ◽

Nigel M. Hooper

Keyword(s):

Amyloid Precursor Protein ◽

Amyloid Β ◽

Proteolytic Processing ◽

Precursor Protein ◽

Aβ Peptides ◽

App Processing ◽

Cellular Expression ◽

Aβ Generation ◽

Human Neuronal Cells ◽

Inducible Cell Line

Abstract Alzheimer’s disease (AD) is a neurodegenerative condition, of which one of the cardinal pathological hallmarks is the extracellular accumulation of amyloid β (Aβ) peptides. These peptides are generated via proteolysis of the amyloid precursor protein (APP), in a manner dependent on the β-secretase, BACE1 and the multicomponent γ-secretase complex. Recent data also suggest a contributory role in AD of transactive response DNA binding protein 43 (TDP-43). There is little insight into a possible mechanism linking TDP-43 and APP processing. To this end, we used cultured human neuronal cells to investigate the ability of TDP-43 to interact with APP and modulate its proteolytic processing. Immunocytochemistry showed TDP-43 to be spatially segregated from both the extranuclear APP holoprotein and its nuclear C-terminal fragment. The latter (APP intracellular domain) was shown to predominantly localise to nucleoli, from which TDP-43 was excluded. Furthermore, neither overexpression of each of the APP isoforms nor siRNA-mediated knockdown of APP had any effect on TDP-43 expression. Doxycycline-stimulated overexpression of TDP-43 was explored in an inducible cell line. Overexpression of TDP-43 had no effect on expression of the APP holoprotein, nor any of the key proteins involved in its proteolysis. Furthermore, increased TDP-43 expression had no effect on BACE1 enzymatic activity or immunoreactivity of Aβ1-40, Aβ1-42 or the Aβ1-40:Aβ1-42 ratio. Also, siRNA-mediated knockdown of TDP-43 had no effect on BACE1 immunoreactivity. Taken together, these data indicate that TDP-43 function and/or dysfunction in AD is likely independent from dysregulation of APP expression and proteolytic processing and Aβ generation.

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Modulation of Aβ generation by small ubiquitin-like modifiers does not require conjugation to target proteins

Biochemical Journal ◽

10.1042/bj20061451 ◽

2007 ◽

Vol 404 (2) ◽

pp. 309-316 ◽

Cited By ~ 47

Author(s):

Véronique Dorval ◽

Matthew J. Mazzella ◽

Paul M. Mathews ◽

Ronald T. Hay ◽

Paul E. Fraser

Keyword(s):

Protein Interactions ◽

Amyloid Β ◽

Specific Effect ◽

Covalent Attachment ◽

Amyloid Β Peptide ◽

Chain Formation ◽

Aβ Peptides ◽

App Processing ◽

Target Proteins ◽

Aβ Amyloid

The sequential processing of the APP (amyloid precursor protein) by the β- and γ-secretase and generation of the Aβ (amyloid-β) peptide is a primary pathological factor in AD (Alzheimer's disease). Regulation of the processing or turnover of these proteins represents potential targets for the development of AD therapies. Sumoylation is a process by which SUMOs (small ubiquitin-like modifiers) are covalently conjugated to target proteins, resulting in a number of functional consequences. These include regulation of protein–protein interactions, intracellular trafficking and protein stability, which all have the potential to impact on several aspects of the amyloidogenic pathway. The present study examines the effects of overexpression and knockdown of the major SUMO isoforms (SUMO1, 2 and 3) on APP processing and the production of Aβ peptides. SUMO3 overexpression significantly increased Aβ40 and Aβ42 secretion, which was accompanied by an increase in full-length APP and its C-terminal fragments. These effects of SUMO3 were independent of its covalent attachment or chain formation, as mutants lacking the motifs responsible for SUMO chain formation or SUMO conjugation led to similar changes in Aβ. SUMO3 overexpression also up-regulated the expression of the transmembrane protease BACE (β-amyloid-cleaving enzyme), but failed to affect levels of several other unrelated proteins. Suppression of SUMO1 or combined SUMO2+3 by RNA interference did not affect APP levels or Aβ production. These findings confirm a specific effect of SUMO3 overexpression on APP processing and the production of Aβ peptides but also suggest that endogenous sumoylation is not essential and likely plays an indirect role in modulating the amyloid processing pathway.

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Mutations in the juxtamembrane segment of the cholesterol-binding site of APP alter its processing and promotes production of shorter, less toxic Aβ peptides

10.1101/2020.11.16.384891 ◽

2020 ◽

Author(s):

Linda Hanbouch ◽

Béatrice Schaack ◽

Amal Kasri ◽

Gaëlle Fontaine ◽

Eleni Gkanatsiou ◽

...

Keyword(s):

Mass Spectrometry ◽

Binding Site ◽

Amyloid Β ◽

Neuronal Cultures ◽

Membrane Cholesterol ◽

Aβ Peptides ◽

App Processing ◽

Juxtamembrane Region ◽

Early Endosomes ◽

Cholesterol Binding

AbstractBackgroundThe brains of patients with Alzheimer’s disease (AD) reveal increased cellular membrane levels of cholesterol. Correspondingly, we previously showed that elevating levels of membrane cholesterol in neuronal cultures recapitulates early AD phenotypes including excessive cleavage of amyloid β (Aβ) peptides from the amyloid precursor protein (APP). Here we aimed to evaluate how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of APP regulates its processing.MethodsWe generated seven single and two double APP mutants at amino acid positions 22, 26, 28, 29, 33, 39 of the Aβ sequence changing the charge and/or hydrophobicity of the targeted amino acids. HEK293T cells were transfected with APP constructs and secreted Aβ peptides were measured using ELISA and mass spectrometry (MS). APP processing in normal and high cholesterol condition, and endocytosis were assessed in stably expressing APPwt and APPK28A HEK293T clones. Finally, we measured the binding of synthetic peptides derived from the Aβ sequence to cholesterol-rich exosomes purified from control HEK293T cells.ResultsMost mutations triggered a reduction in the production of Aβ40 and Aβ42 peptides, whereas only juxtamembrane mutants resulted in the generation of shorter Aβ peptides. We confirmed by mass spectrometry this specific change in the profile of secreted Aβ peptides for the most characteristic APPK28A mutant. A transient increase of plasma membrane cholesterol enhanced the production of Aβ40 by APPWT, an effect absent with APPK28A. The enzymatic activity of α-, β- and γ-secretases remained unchanged in cells expressing APPK28A. Similarly, APPK28A subcellular localization in early endosomes did not differ to APPWT. Finally, WT but not CBS mutant Aβ derived peptides bound to cholesterol-rich exosomes.ConclusionsTaken together, these data reveal a major role of the juxtamembrane region of APP in binding to cholesterol and accordingly in the regulation of APP processing. Binding of cholesterol to K28 could staple APP to the juxtamembrane region thereby permitting access to γ-secretase cleavage at positions 40-42. The APPK28 mutant would lie deeper in the membrane, facilitating the production of shorter Aβ peptides and unveiling this specific region as a novel target for reducing the production of toxic Aβ species.

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Rab35 and glucocorticoids regulate APP and BACE1 trafficking to modulate Aβ production

Cell Death and Disease ◽

10.1038/s41419-021-04433-w ◽

2021 ◽

Vol 12 (12) ◽

Author(s):

Viktoriya Zhuravleva ◽

João Vaz-Silva ◽

Mei Zhu ◽

Patricia Gomes ◽

Joana M. Silva ◽

...

Keyword(s):

Tau Protein ◽

Intracellular Trafficking ◽

Stress Hormones ◽

Amyloid Β ◽

Small Gtpase ◽

Aβ Peptides ◽

App Processing ◽

Hyperphosphorylated Tau Protein ◽

Open Question ◽

Aβ Production

AbstractChronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer’s disease (AD) and promote AD pathomechanisms, including overproduction of toxic amyloid-β (Aβ) peptides and intraneuronal accumulation of hyperphosphorylated Tau protein. The latter is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in Aβ overproduction remains an open question. Here, we find that hippocampal Rab35 levels are decreased not only by stress/GC but also by aging, another AD risk factor. Moreover, we show that Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking steps for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Finally, we demonstrate that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by high GC levels. These studies identify Rab35 as a key regulator of APP processing and suggest that its downregulation may contribute to stress-related and AD-related amyloidogenesis.

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Rab35 and glucocorticoids regulate APP and BACE1 trafficking to modulate Aβ production

10.1101/2021.05.10.443354 ◽

2021 ◽

Author(s):

Viktoriya Zhuravleva ◽

João Vaz Silva ◽

Mei Zhu ◽

Patrícia Gomes ◽

Joana M Silva ◽

...

Keyword(s):

Tau Protein ◽

Intracellular Trafficking ◽

Stress Hormones ◽

Amyloid Β ◽

Small Gtpase ◽

Aβ Peptides ◽

App Processing ◽

Hyperphosphorylated Tau Protein ◽

Open Question ◽

Aβ Production

Chronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer's disease (AD) and promote AD pathomechanisms, including overproduction of toxic amyloid-β (Aβ) peptides and intraneuronal accumulation of hyperphosphorylated Tau protein. The latter is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in Aβ overproduction remains an open question. Here, we find that hippocampal Rab35 levels are decreased not only by stress/GC but also by aging, another AD risk factor. Moreover, Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking events for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Finally, we show that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by high GC levels. These studies identify Rab35 as a key regulator of APP processing and suggest that its downregulation may contribute to stress- and AD-related amyloidogenesis.

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Contribution of the endosomal-lysosomal and proteasomal systems in Amyloid-β Precursor Protein derived fragments processing

10.1101/300921 ◽

2018 ◽

Cited By ~ 1

Author(s):

Caroline Evrard ◽

Pascal Kienlen-Campard ◽

Rémi Opsomer ◽

Bernadette Tasiaux ◽

Jean-Noël Octave ◽

...

Keyword(s):

Amyloid Β ◽

Full Length ◽

Precursor Protein ◽

Degradation Pathways ◽

Aβ Peptides ◽

Hek 293 ◽

App Processing ◽

Amyloid Deposits ◽

App Metabolism ◽

Carboxy Terminal

AbstractAβ peptides, the major components of amyloid deposits of Alzheimer’s disease, are released following sequential cleavages by secretases of its precursor named the amyloid precursor protein (APP). In addition to secretases, degradation pathways, in particular the endosomal/lysosomal and proteasomal systems have also been reported to contribute to APP processing. However, the respective role of each of these pathways towards APP metabolism remains to be established. To address this, we used HEK 293 cells and primary neurons expressing full-length APPWT or the β-secretase-derived C99 fragments (β-CTFs) in which degradation pathways were selectively blocked using pharmacological drugs. APP metabolites, including carboxy-terminal fragments (CTFs), soluble APP (sAPP) and Aβ peptides were studied. In this report, we show that APP-CTFs produced from endogenous or overexpressed full-length APP are mainly processed by γ-secretase and the endosomal/lysosomal pathway, while in sharp contrast, overexpressed C99 alone is mainly degraded by the proteasome and to a lesser extent by γ-secretase.

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Accelerated neuronal and synaptic maturation by BrainPhys medium increases Aβ secretion and alters Aβ peptide ratios from iPSC-derived cortical neurons

Scientific Reports ◽

10.1038/s41598-020-57516-7 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Tugce Munise Satir ◽

Faisal Hayat Nazir ◽

Dzeneta Vizlin-Hodzic ◽

Erik Hardselius ◽

Kaj Blennow ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Cortical Neurons ◽

Amyloid Β ◽

Induced Pluripotent Stem Cell ◽

Synaptic Function ◽

Neuronal Maturation ◽

Aβ Peptides ◽

App Processing ◽

Synaptic Maturation ◽

Induced Pluripotent

AbstractOne of the neuropathological hallmarks of Alzheimer’s disease (AD) is cerebral deposition of amyloid plaques composed of amyloid β (Aβ) peptides and the cerebrospinal fluid concentrations of those peptides are used as a biomarker for AD. Mature induced pluripotent stem cell (iPSC)-derived cortical neurons secrete Aβ peptides in ratios comparable to those secreted to cerebrospinal fluid in human, however the protocol to achieve mature neurons is time consuming. In this study, we investigated if differentiation of neuroprogenitor cells (NPCs) in BrainPhys medium, previously reported to enhance synaptic function of neurons in culture, would accelerate neuronal maturation and, thus increase Aβ secretion as compared to the conventional neural maintenance medium. We found that NPCs cultured in BrainPhys displayed increased expression of markers for cortical deep-layer neurons, increased synaptic maturation and number of astroglial cells. This accelerated neuronal maturation was accompanied by increased APP processing, resulting in increased secretion of Aβ peptides and an increased Aβ38 to Aβ40 and Aβ42 ratio. However, during long-term culturing in BrainPhys, non-neuronal cells appeared and eventually took over the cultures. Taken together, BrainPhys culturing accelerated neuronal maturation and increased Aβ secretion from iPSC-derived cortical neurons, but changed the cellular composition of the cultures.

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