p75 Neurotrophin Receptor Cleavage by α- and γ-Secretases Is Required for Neurotrophin-mediated Proliferation of Brain Tumor-initiating Cells

Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.     

Secretion Stimulates Intramembrane Proteolysis of a Secretory Granule Membrane Enzyme

Regulated intramembrane proteolysis, a highly conserved process employed by diverse regulatory pathways, can release soluble fragments that directly or indirectly modulate gene expression. In this study we used pharmacological tools to identify peptidylglycine α-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a γ-secretase substrate. PAM, an essential enzyme, catalyzes the final step in the synthesis of the majority of neuropeptides that control metabolic homeostasis. Mass spectroscopy was most consistent with the presence of multiple closely spaced NH₂ termini, suggesting that cleavage occurred near the middle of the PAM transmembrane domain. The luminal domains of PAM must undergo a series of prohormone convertase or α-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a γ-secretase-like cleavage. Cleavage by γ-secretase generates a soluble fragment of the cytosolic domain (sf-CD) that is known to localize to the nucleus. Although PAM sf-CD is unstable in AtT-20 corticotroph tumor cells, it is readily detected in primary rat anterior pituitary cells. PAM isoform expression, which is tissue-specific and developmentally regulated, affects the efficiency with which sf-CD is produced. sf-CD levels are also modulated by the phosphorylation status of the cytosolic domain and by the ability of the cytosolic domain to interact with cytosolic proteins. sf-CD is produced by primary rat anterior pituitary cells in response to secretogogue, suggesting that sf-CD acts as a signaling molecule relaying information about secretion from the secretory granule to the nucleus.     

γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

Background

γ-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid β-peptide (Aβ) and the APP intracellular domain. Aβ is widely believed to have a causative role in Alzheimer''s disease pathogenesis, and therefore modulation of γ-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of γ-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to γ-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs).

Principal Findings

Here, we investigated γ-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Aβ40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Aβ were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Aβ40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased.

Conclusions

In summary our data suggests that γ-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of γ-secretase inhibitors.     

The Effects of Transmembrane Sequence and Dimerization on Cleavage of the p75 Neurotrophin Receptor by γ-Secretase

Cleavage of transmembrane receptors by γ-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function. Although relatively little is known about the molecular mechanism of γ-secretase enzymatic activity, it is becoming clear that substrate dimerization and/or the α-helical structure of the substrate can regulate the site and rate of γ-secretase activity. Here we show that the transmembrane domain of the pan-neurotrophin receptor p75^NTR, best known for regulating neuronal death, is sufficient for its homodimerization. Although the p75^NTR ligands NGF and pro-NGF do not induce homerdimerization or RIP, homodimers of p75^NTR are γ-secretase substrates. However, dimerization is not a requirement for p75^NTR cleavage, suggesting that γ-secretase has the ability to recognize and cleave each receptor molecule independently. The transmembrane cysteine 257, which mediates covalent p75^NTR interactions, is not crucial for homodimerization, but this residue is required for normal rates of γ-secretase cleavage. Similarly, mutation of the residues alanine 262 and glycine 266 of an AXXXG dimerization motif flanking the γ-secretase cleavage site within the p75^NTR transmembrane domain alters the orientation of the domain and inhibits γ-secretase cleavage of p75^NTR. Nonetheless, heteromer interactions of p75^NTR with TrkA increase full-length p75^NTR homodimerization, which in turn potentiates the rate of γ-cleavage following TrkA activation independently of rates of α-cleavage. These results provide support for the idea that the helical structure of the p75^NTR transmembrane domain, which may be affected by co-receptor interactions, is a key element in γ-secretase-catalyzed cleavage.     

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating ��-Secretase Complex Assembly and Dictating the Amyloid �� Peptide (A��) Profile without Affecting Notch Processing

γ-Secretase is an enzyme complex that mediates both Notch signaling and β-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid β peptide (Aβ), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of γ-secretase gives rise to Aβ peptides of different lengths, where Aβ42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320–374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of γ-secretase, for γ-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, γ-secretase complex formation, and had a differential effect on Aβ-peptide production. Although the production of Aβ38, Aβ39, and Aβ40 was severely impaired, the effect on Aβ42 was affected to a lesser extent, implying that the production of the AD-related Aβ42 peptide is separate from the production of the Aβ38, Aβ39, and Aβ40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ϵ/S3 and γ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Aβ peptides without affecting Notch processing, two parameters of significance when considering γ-secretase as a target for pharmaceutical intervention in AD.     

Alcadein Cleavages by Amyloid ��-Precursor Protein (APP) ��- and ��-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ��-Secretase Dysfunction

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc_α, Alc_β, and Alc_γ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc_α, p3-Alc_β, and p3-Alc_γ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc_α, p3-Alc_β, and p3-Alc_γ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.     

Activity-dependent α-Cleavage of Nectin-1 Is Mediated by A Disintegrin and Metalloprotease 10 (ADAM10)

Nectin-1 is known to undergo ectodomain shedding by α-secretase and subsequent proteolytic processing by γ-secretase. How secretase-mediated cleavage of nectin-1 is regulated in neuronal cells and how nectin-1 cleavage affects synaptic adhesion is poorly understood. We have investigated α-and γ-secretase-mediated processing of nectin-1 in primary cortical neurons and identified which protease acts as a α-secretase. We report here that NMDA receptor activation, but not stimulation of AMPA or metabotropic glutamate receptors, resulted in robust α- and γ-secretase cleavage of nectin-1 in mature cortical neurons. Cleavage of nectin-1 required influx of Ca²⁺ through the NMDA receptor, and activation of calmodulin, but was not dependent on calcium/calmodulin-dependent protein kinase II (CaMKII) activation. We found that ADAM10 is the major secretase responsible for nectin-1 ectodomain cleavage in neurons and the brain. These observations suggest that α- and γ-secretase processing of nectin-1 is a Ca²⁺/calmodulin-regulated event that occurs under conditions of activity-dependent synaptic plasticity and ADAM10 and γ-secretase are responsible for these cleavage events.     

Cyclopamine Modulates γ-Secretase-mediated Cleavage of Amyloid Precursor Protein by Altering Its Subcellular Trafficking and Lysosomal Degradation

Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-β (Aβ), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aβ is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aβ generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aβ generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.     

Cytoplasmic Fragment of Alcadein α Generated by Regulated Intramembrane Proteolysis Enhances Amyloid β-Protein Precursor (APP) Transport into the Late Secretory Pathway and Facilitates APP Cleavage

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo.     

Lysine 63-linked Polyubiquitination of TAK1 at Lysine 158 Is Required for Tumor Necrosis Factor ��- and Interleukin-1��-induced IKK/NF-��B and JNK/AP-1 Activation

Transforming growth factor-β-activated kinase 1 (TAK1) plays an essential role in the tumor necrosis factor α (TNFα)- and interleukin-1β (IL-1β)-induced IκB kinase (IKK)/nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) activation. Here we report that TNFα and IL-1β induce Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue within the kinase domain. Tumor necrosis factor receptor-associated factors 2 and 6 (TRAF2 and -6) act as the ubiquitin E3 ligases to mediate Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue in vivo and in vitro. Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue is required for TAK1-mediated IKK complex recruitment. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild type or a TAK1 mutant containing a K158R mutation revealed the importance of this site in TNFα and IL-1β-mediated IKK/NF-κB and JNK/AP-1 activation as well as IL-6 gene expression. Our findings demonstrate that Lys⁶³-linked polyubiquitination of TAK1 at Lys¹⁵⁸ is essential for its own kinase activation and its ability to mediate its downstream signal transduction pathways in response to TNFα and IL-1β stimulation.     

β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators

Pathogenic generation of the 42-amino acid variant of the amyloid β-peptide (Aβ) by β- and γ-secretase cleavage of the β-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Aβ₄₂ production by γ-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Aβ domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of γ-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Aβ domain regarding their response to GSMs. We found that Aβ₄₂-increasing familial AD mutations of the γ-secretase cleavage site domain responded robustly to Aβ₄₂-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Aβ₄₂ produced. We thus expect that familial AD patients carrying mutations at the γ-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other Aβ species (Aβ₄₁, Aβ₃₉) could also be lowered besides Aβ₄₂. Moreover, certain mutations simultaneously increased Aβ₄₂ and the shorter peptide Aβ₃₈, arguing that the proposed precursor-product relationship of these Aβ species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Aβ₄₂ generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.     

Interplay between α-, β-, and γ-Secretases Determines Biphasic Amyloid-β Protein Level in the Presence of a γ-Secretase Inhibitor

Amyloid-β (Aβ) is produced by the consecutive cleavage of amyloid precursor protein (APP) first by β-secretase, generating C99, and then by γ-secretase. APP is also cleaved by α-secretase. It is hypothesized that reducing the production of Aβ in the brain may slow the progression of Alzheimer disease. Therefore, different γ-secretase inhibitors have been developed to reduce Aβ production. Paradoxically, it has been shown that low to moderate inhibitor concentrations cause a rise in Aβ production in different cell lines, in different animal models, and also in humans. A mechanistic understanding of the Aβ rise remains elusive. Here, a minimal mathematical model has been developed that quantitatively describes the Aβ dynamics in cell lines that exhibit the rise as well as in cell lines that do not. The model includes steps of APP processing through both the so-called amyloidogenic pathway and the so-called non-amyloidogenic pathway. It is shown that the cross-talk between these two pathways accounts for the increase in Aβ production in response to inhibitor, i.e. an increase in C99 will inhibit the non-amyloidogenic pathway, redirecting APP to be cleaved by β-secretase, leading to an additional increase in C99 that overcomes the loss in γ-secretase activity. With a minor extension, the model also describes plasma Aβ profiles observed in humans upon dosing with a γ-secretase inhibitor. In conclusion, this mechanistic model rationalizes a series of experimental results that spans from in vitro to in vivo and to humans. This has important implications for the development of drugs targeting Aβ production in Alzheimer disease.     

The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates’ ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.     

Prevention of CCAAT/Enhancer-binding Protein �� DNA Binding by Hypoxia during Adipogenesis

Upon exposure to adipogenesis-inducing hormones, confluent 3T3-L1 preadipocytes express C/EBPβ (CCAAT/enhancer binding protein β). Early induced C/EBPβ is inactive but, after a lag period, acquires its DNA-binding capability by sequential phosphorylation. During this period, preadipocytes pass the G₁/S checkpoint synchronously. Thr¹⁸⁸ of C/EBPβ is phosphorylated initially to prime the factor for subsequent phosphorylation at Ser¹⁸⁴ or Thr¹⁷⁹ by GSK3β, which translocates into the nuclei during the G₁/S transition. Many events take place during the G₁/S transition, including reduction in p27^Kip1 protein levels, retinoblastoma (Rb) phosphorylation, GSK3β nuclear translocation, and C/EBPβ binding to target promoters. During hypoxia, hypoxia-inducible factor-1α (HIF-1α) is stabilized, thus maintaining expression of p27^Kip1, which inhibits Rb phosphorylation. Even under normoxic conditions, constitutive expression of p27^Kip1 blocks the nuclear translocation of GSK3β and DNA binding capability of C/EBPβ. Hypoxia also blocks nuclear translocation of GSK3β and DNA binding capability of C/EBPβ in HIF-1α knockdown 3T3-L1 cells that fail to induce p27^Kip1. Nonetheless, under hypoxia, these cells can block Rb phosphorylation and the G₁/S transition. Altogether, these findings suggest that hypoxia prevents the nuclear translocation of GSK3β and the DNA binding capability of C/EBPβ by blocking the G₁/S transition through HIF-1α-dependent induction of p27^Kip1 and an HIF-1α/p27-independent mechanism.     

Proline cis/trans-Isomerase Pin1 Regulates Peroxisome Proliferator-activated Receptor �� Activity through the Direct Binding to the Activation Function-1 Domain

The important roles of a nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPARγ activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPARγ activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPARγ. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPARγ-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPARγ.     

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.