Content uploaded by Fabien Llambi
Author content
All content in this area was uploaded by Fabien Llambi
Content may be subject to copyright.
A preview of the PDF is not available
Netrin-1 interacts with amyloid precursor protein and regulates amyloid-?? production
Abstract and Figures
The beta-amyloid precursor protein (APP) is an orphan transmembrane receptor whose physiological role is largely unknown. APP is cleaved by proteases generating amyloid-beta (Abeta) peptide, the main component of the amyloid plaques that are associated with Alzheimer's disease. Here, we show that APP binds netrin-1, a multifunctional guidance and trophic factor. Netrin-1 binding modulates APP signaling triggering APP intracellular domain (AICD)-dependent gene transcription. Furthermore, netrin-1 binding suppresses Abeta peptide production in brain slices from Alzheimer model transgenic mice. In this mouse model, decreased netrin-1 expression is associated with increased Abeta concentration, thus supporting netrin-1 as a key regulator of Abeta production. Finally, we show that netrin-1 brain administration in Alzheimer model transgenic mice may be associated with an amelioration of the Alzheimer's phenotype.
Figures - uploaded by Fabien Llambi
Author content
All figure content in this area was uploaded by Fabien Llambi
Content may be subject to copyright.
... The Netrin-1/DCC complex co-immunoprecipitates with APLP1, which led to identification of APP as a functional Netrin-1 receptor [59]. Furthermore, administration of Netrin-1 was found to decrease Aβ levels [59]. ...
... The Netrin-1/DCC complex co-immunoprecipitates with APLP1, which led to identification of APP as a functional Netrin-1 receptor [59]. Furthermore, administration of Netrin-1 was found to decrease Aβ levels [59]. This further supports a link between BACE1 and DCC. ...
... As the DCC/Netrin-1 interaction has been shown to play a role in the adult brain in axon guidance and angiogenesis, this may contribute to the neuronal and vascular impairments observed in AD. As Netrin-1 has been reported to have a similar affinity for APP, as DCC, increased BACE1 activity may lead to increased availability of Netrin-1, and therefore increased binding to APP [59]. Netrin-1 binding to APP has been shown to induce APP mediated signalling and gene transcription and suppress Aβ production. ...
The β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer’s disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aβ) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aβ concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD.
... Fourteen proteins overlapped between all three tissues in the context of AD (Fig. 6a) (Supplementary Table 19). Many of these proteins have established links or are highly relevant to LOAD, such as Spondin 1 (SPON1), involved in the processing of amyloid precursor protein (APP) 39 ; Secreted Modular Calcium-Binding Protein 1 (SMOC1) previously proposed as a biomarker of LOAD in postmortem brains and CSF 40 ; Netrin-1 (NTN1), an interactor of APP and regulator of amyloid-beta production 41 ; Neuro lament light (NEFL), previously proposed as a plasma biomarker for LOAD and axon injury 42,43 and Von Willebrand factor (VWF), known for its role in blood clotting and associations with LOAD 44 (Supplementary Table 19). Notably, some of the APOE-dependent proteins were associated with AD across all three tissues such as TBCA and TP53I11. ...
The current demand for early intervention, prevention, and treatment of late onset Alzheimer’s disease (LOAD) warrants deeper understanding of the underlying molecular processes which could contribute to biomarker and drug target discovery. Utilizing high-throughput proteomic measurements in serum from a prospective population-based cohort of older adults (n = 5,294), we identified 303 unique proteins associated with incident LOAD (median follow-up 12.8 years). Over 40% of these proteins were associated with LOAD independently of APOE- ε 4 carrier status. These proteins were implicated in neuronal processes and overlapped with protein signatures of LOAD in brain and cerebrospinal fluid. We found 17 proteins which LOAD-association was strongly dependent on APOE- ε 4 carrier status. Most of them showed consistent associations with LOAD in cerebrospinal fluid and a third had brain-specific gene expression. Remarkably, four proteins in this group (TBCA, ARL2, S100A13 and IRF6) were downregulated by APOE- ε 4 yet upregulated as a consequence of LOAD as determined in a bi-directional Mendelian randomization analysis, reflecting a potential response to the disease onset. Accordingly, the direct association of these proteins to LOAD was reversed upon APOE- ε 4 genotype adjustment, a finding which we replicate in an external cohort (n = 719). Our findings provide an insight into the dysregulated pathways that may lead to the development and early detection of LOAD, including those both independent and dependent on APOE- ε 4 . Importantly, many of the LOAD-associated proteins we find in the circulation have been found to be expressed - and have a direct link with AD - in brain tissue. Thus, the proteins identified here, and their upstream modulating pathways, provide a new source of circulating biomarker and therapeutic target candidates for LOAD.
... This is not surprising given the contribution of Netrin-1 to synaptic function, which is involved in learning and memory [33,45]. One way that Netrin-1 may protect against Alzheimer's is by negatively regulating amyloid peptide [46]. Since Alzheimer's patients appear to have reduced melanoma incidence [47], the low Netrin-1 expression and high amyloid beta levels produced in Alzheimer's brains could represent a possible protective factor against melanoma. ...
Netrin-1 is a neural guidance factor that regulates migration and positioning of neural crest-derived cells during embryonic development. Depending on the type of Netrin-1 receptor expression, cells are either attracted or repulsed by Netrin-1. Postnatal expression of Netrin-1 is detected in brain, colon, liver, and kidney, which are common sites of cancer metastasis, including melanoma. Thus, understanding the dynamics between Netrin-1 and its receptors could explain the attraction of melanoma towards these Netrin-1-expressing tissues. Here, we investigate whether the Netrin-1-attractive receptor Neogenin can affect migration of melanoma cells towards a Netrin-1 source. Results from Western blot (WB) analysis show higher expression of Neogenin in aggressive compared to non-aggressive melanoma cells. Cell migration experiments show increased migration of Neogenin-expressing aggressive melanoma cells towards exogenous, soluble recombinant human Netrin-1 and towards a Netrin-1-expressing cell line. Furthermore, WB reveals ERK1/2 activation and increased N-cadherin expression in Neogenin-expressing aggressive melanoma cells treated with rhNetrin-1. Moreover, treatment with anti-Neogenin blocking antibody caused decreased migration towards Netrin-1-expressing cells and reduced ERK1/2 activity in Neogenin-expressing aggressive melanoma cells. These results suggest Neogenin may play a role during migration of melanoma cells towards Netrin-1 via ERK1/2 signaling.
... Bu farelerin beyinlerine netrin-1 eklenmesinin Alzheimer fenotipinde iyileşmeye neden olduğu gösterilmiştir. 24 Sun ve ark. ise çalışmalarında, deneysel AH olan farelerin hem serebrospinal sıvılarında hem de serumlarında netrin-1 düzeyinin sağlıklı farelere göre düşük olduğunu bildirmişlerdir. ...
... In addition, it was reported that UNC5C, a netrin-1 receptor, would also be cleaved by δ-secretase [27]. UNC5C could inhibit the production of Aβ and alleviate the AD pathologies after binding with ligand [28,29]. The hypothesis of δsecretase has been proved in mice models, as the AD pathologies such as cognitive dysfunctions were weakened by inhibiting the expression of δ-secretase. ...
The amyloid cascade hypothesis has always been a research focus in the therapeutic field of Alzheimer's disease (AD) since it was put forward. Numerous researchers attempted to find drugs for AD treatment based on this hypothesis. To promote the research of anti-AD drugs development, the current hypothesis and pathogenesis were reviewed with expounding of β-amyloid generation from its precursor protein and related transformations. Meanwhile, the present drug development strategies aimed at each stage in this hypothesis were also summarized. Several strategies especially immunotherapy showed the optimistic results in clinical trials, but only a small percentage of them eventually succeeded. In this review, we also tried to point out some common problems of drug development in preclinical and clinical studies which might be settled through multidisciplinary cooperation as well as the understanding that reinforces the amyloid cascade hypothesis.
Netrin-1 was initially discovered as a neuronal growth cue for axonal guidance, and its functions have later been identified in inflammation, tumorigenesis, neurodegeneration, and other disorders. We have recently found its alterations in the brains with Alzheimer’s disease, which might provide important clues to the mechanisms of some unique pathologies. To provide better understanding of this promising molecule, we here summarize research progresses in genetics, pathology, biochemistry, cell biology and other studies of Netrin-1 about its mechanistic roles and biomarker potentials with an emphasis on clinical neurodegenerative disorders in order to expand understanding of this promising molecular player in human diseases.
Neurodegenerative diseases are characterized by the progressive loss of structure or function of neurons. In this Spotlight, we explore the idea that genetic forms of neurodegenerative disorders might be rooted in neural development. Focusing on Alzheimer's, Parkinson's and Huntington's disease, we first provide a brief overview of the pathology for these diseases. Although neurodegenerative diseases are generally thought of as late-onset diseases, we discuss recent evidence promoting the notion that they might be considered neurodevelopmental disorders. With this view in mind, we consider the suitability of animal models for studying these diseases, highlighting human-specific features of human brain development. We conclude by proposing that one such feature, human-specific regulation of neurogenic time, might be key to understanding the etiology and pathophysiology of human neurodegenerative disease.
Hallmark pathological features of brain trauma are axonal degeneration and demyelination because myelin-producing oligodendrocytes (OLs) are particularly vulnerable to injury-induced death signals. To reveal mechanisms responsible for this OL loss, we examined a novel class of “death receptors” called dependence receptors (DepRs). DepRs initiate pro-death signals in the absence of their respective ligand(s), yet little is known about their role after injury. Here, we investigated whether the deleted in colorectal cancer (DCC) DepR contributes to OL loss after brain injury. We found that administration of its netrin-1 ligand is sufficient to block OL cell death. We also show that upon acute injury, DCC is upregulated while netrin-1 is downregulated in perilesional tissues. Moreover, after genetically silencing pro-death activity using DCCD1290N mutant mice, we observed greater OL survival, greater myelin integrity, and improved motor function. Our findings uncover a novel role for the netrin-1/DCC pathway in regulating OL loss in the traumatically injured brain.
A critical challenge in current research of Alzheimer’s disease (AD) is to clarify the relationship between early neuropathology and network dysfunction. In the present work, the new generation AppNL-FxMAPT double knock in (dKI) model was used to evaluate early stages of AD. The initial step of tau pathology was restricted to the perirhinal-entorhinal region, sparing the hippocampus. This discrete neuropathological sign coincided with deficits in object-place associative memory, one of the earliest recognition memory forms affected in individuals at risk for developing AD. Analyses of task-dependent c-Fos activation were carried out in regions susceptible to early AD pathology, and revealed decreased network efficiency during memory retrieval, likely due to reduced information flow through the retrosplenial cortex. Our results suggest that early perirhinal-entorhinal tau pathology is associated with local hyper-activity which spreads towards connected regions such as the claustrum, the medial prefrontal cortex and ultimately the key retrosplenial hub which is needed to relay information flow from frontal to temporal lobes.
The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2 ⁻ /− /APLP1 ⁻ /− and APLP2 ⁻ /− /APP ⁻ /− mice proved lethal early postnatally. Surprisingly, APLP1 ⁻ /− /APP ⁻ /− mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1 ⁻ /− /APP ⁻ /− /APLP2 +/− mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicity in vitro .
Apolipoprotein E, α2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer’s disease. All three proteins
are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that
has also been genetically linked to Alzheimer’s disease. The cytoplasmic tails of LRP and other members of the LDL receptor
gene family contain NPxY motifs that are required for receptor endocytosis. To investigate whether these receptors may have
functions that go beyond ligand internalization, e.g. possible roles in cellular signaling, we searched for proteins that might interact with the cytoplasmic tails of the receptors.
A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been
described. Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, LDL receptor,
and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes. FE65
contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that
LRP can modulate the intracellular trafficking of APP. Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor
tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular
pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal.
The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence
of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium
from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican bound strongly to the APP affinity column. Purified glypican bound to APP with an equilibrium dissociation
constant of 2.8 nM and inhibited APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican was
specific for APP, as glypican did not inhibit laminin-induced neurite outgrowth. Furthermore, treatment of cultures with 4-methylumbelliferyl-β-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibited APP-induced neurite outgrowth but did not inhibit
laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP
to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP.
Progressive cerebral deposition of extracellular filaments composed of the beta-amyloid protein (beta AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the beta AP precursor (beta APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated beta AP deposition. Using two antibodies to the predicted carboxyl terminus of beta APP, we have identified the native beta APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundant in fetal brain, and detected in nonneural tissues that contain beta APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. Confirmation that the immunodetected tissue proteins are forms of beta APP was obtained when mammalian cells transfected with a full-length beta APP cDNA showed selectively augmented expression of 110- to 135-kDa proteins and specific immunocytochemical staining. Unexpectedly, the antibodies to the carboxyl terminus of beta APP labeled amyloid-containing senile plaques in AD brain. We conclude that the highly conserved beta APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of approximately 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the beta APP into insoluble filaments occurs locally in cortical regions that develop beta-amyloid deposits with age.
Transgenic FVB/N mice overexpressing human (Hu) or mouse (Mo) Alzheimer amyloid precursor protein (APP695) die early and develop a CNS disorder that includes neophobia and impaired spatial alternation, with diminished glucose utilization and astrogliosis mainly in the cerebrum. Age at onset of neophobia and age at death decrease with increasing levels of brain APP. HuAPP transgenes induce death much earlier than MoAPP transgenes expressed at similar levels. No extracellular amyloid was detected, indicating that some deleterious processes related to APP overexpression are dissociated from formation of amyloid. A similar clinical syndrome occurs spontaneously in approximately 20% of nontransgenic mice when they reach mid- to late-adult life, suggesting that APP overexpression may accelerate a naturally occurring age-related CNS disorder in FVB/N mice.
In vertebrates, commissural axons pioneer a circumferential pathway to the floor plate at the ventral midline of the embryonic spinal cord. Floor plate cells secrete a diffusible factor that promotes the outgrowth of commissural axons in vitro. We have purified from embryonic chick brain two proteins, netrin-1 and netrin-2, that each possess commissural axon outgrowth-promoting activity, and we have also identified a distinct activity that potentiates their effects. Cloning of cDNAs encoding the two netrins shows that they are homologous to UNC-6, a laminin-related protein required for the circumferential migration of cells and axons in C. elegans. This homology suggests that growth cones in the vertebrate spinal cord and the nematode are responsive to similar molecular cues.
The amyloid protein precursor (APP) of Alzheimer's disease is synthesized as an integral transmembrane protein that is released from cells in culture following proteolytic cleavage. The function of released APP is not known, although there is evidence that the protein may bind to components of the extracellular matrix (ECM). In the present study, substratum-bound APP stimulated neurite outgrowth in cultures of chick sympathetic and mouse hippocampal neurons. This effect was dependent upon the presence of substratum-bound heparan sulfate proteoglycans (HSPG). The effect of APP on neurite outgrowth was comparable to that of laminin. A 14 K N-terminal fragment of APP was found to bind heparin and a region close to the N-terminus of APP (residues 96-110) identified as a potential heparin-binding domain based on secondary structure predictions and molecular modeling. Mutagenesis of three basic residues (lysine-99, arginine-100, and arginine-102) resulted in a recombinant protein (APPhep) with decreased heparin-binding capacity. A peptide homologous to the heparin-binding domain was synthesized and found to bind strongly to heparin and to inhibit binding of 125I-labeled APP to heparin (IC50 approximately 10(-7) M). The peptide blocked the effect of APP on neurite outgrowth (IC50 approximately 10(-7) M), whereas two other peptides homologous to other domains in APP had no effect. The results indicate that the binding of APP to HSPG in the ECM may stimulate the effects of APP on neurite outgrowth.
The specific binding of the amyloid precursor protein (APP) to extracellular matrix molecules suggests that APP regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. On the molecular level APP has binding sites for collagen, laminin, and glycosaminoglycans which is a characteristic feature of cell adhesion molecules. We have examined the interactions between the APP and collagen types I and IV and identified the corresponding binding sites on APP and collagen type I.
We show that APP bound most efficiently to collagen type I in a concentration-dependent and specific manner in the native and heat-denatured states, suggesting an involvement of a contiguous binding site on collagen. This binding site was identified on the cyanogen bromide fragment α1(I)CB6 of collagen type I, which also binds heparin. APP did not bind to collagen type I-heparin complexes, which suggests that there are overlapping binding sites for heparin and APP on collagen. We localized the site of APP that mediates collagen binding within residues 448-465 of APP⁶⁹⁵, which are encoded by the ubiquitously expressed APP exon 12, whereas the high affinity heparin binding site of APP is located in exon 9. Since a peptide encompassing this region binds to collagen type I and inhibits APP-collagen type I binding in nanomolar concentrations, this region may comprise the major part of the collagen type I binding site of APP. Moreover, our data also indicate that the collagen binding site is involved in APP-APP interaction that can be modulated by Zn(II) and heparin. Taken together, the data suggest that the regulation of APP binding to collagen type I by heparin occurs through the competitive binding of heparin and APP to collagen.
The physiological functions of the beta-amyloid precursor protein (APP) may include nuclear signaling. To characterize the role of the APP adaptor proteins Fe65, Jip1b, X11alpha (MINT1) and the chromatin-associated protein Tip60, we analyzed their interactions by confocal microscopy and co-immunoprecipitations. AICD corresponding to S3-cleaved APP bound to Fe65 that transported it to nuclei and docked it to Tip60. These proteins formed AICD-Fe65-Tip60 (AFT) complexes that were concentrated in spherical nuclear spots. gamma-Secretase inhibitors prevented AFT-complex formation with AICD derived from full-length APP. The APP adaptor protein Jip1b also transported AICD to nuclei and docked it to Tip60, but AICD-Jip1b-Tip60 (AJT) complexes had different, speckle-like morphology. By contrast, X11alpha trapped AICD in the cytosol. Induced AICD expression identified the APP-effector genes APP, BACE, Tip60, GSK3beta and KAI1, but not the Notch-effector gene Hes1 as transcriptional targets. These data establish a role for APP in nuclear signaling, and they suggest that therapeutic strategies designed to modulate the cleavage of APP affect AICD-dependent signaling.
The guidance of developing axons in the nervous system is mediated partly by diffusible chemoattractants secreted by axonal target cells. Netrins are chemoattractants for commissural axons in the vertebrate spinal cord, but the mechanisms through which they produce their effects are unknown. We show that Deleted in Colorectal Cancer (DCC), a transmembrane protein of the immunoglobulin superfamily, is expressed on spinal commissural axons and possesses netrin-1-binding activity. Moreover, an antibody to DCC selectively blocks the netrin-1-dependent outgrowth of commissural axons in vitro. These results indicate that DCC is a receptor or a component of a receptor that mediates the effects of netrin-1 on commissural axons, and they complement genetic evidence for interactions between DCC and netrin homologs in C. elegans and Drosophila.