The secreted Alzheimer-related amyloid precursor protein fragment has an essential role in C. elegans

Mutations in the gene encoding the amyloid precursor protein (APP) or the enzymes that process APP are correlated with familial Alzheimer disease. Alzheimer disease is also associated with insulin resistance (type 2 diabetes). In our recently published study,¹ we obtained genetic evidence that the extracellular fragment of APL-1, the C. elegans ortholog of human APP, may act as a signaling molecule to modulate insulin and nuclear hormone pathways in C. elegans development. In addition, independent of insulin and nuclear hormone signaling, high levels of the extracellular fragment of APL-1 (sAPL-1) leads to a temperature-sensitive embryonic lethality, which is dependent on activity of a predicted receptor protein tyrosine phosphatase (MOA-1/R155.2). Furthermore, this embryonic lethality is enhanced by knockdown of a predicted prion-like protein (pqn-29). The precise molecular mechanisms underlying these processes remain to be determined. Here, we present hypothetical models as to how sAPL-1 signaling influences metabolic and developmental pathways. Together, with previous findings in mammals that the extracellular domain of mammalian APP (sAPP) binds to a death-receptor,² our findings support the model that sAPP signaling affects critical biological processes.     

Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans

Background

Alzheimer''s disease (AD) is a neurodegenerative disorder primarily characterized by the deposition of β-amyloid plaques in the brain. Plaques are composed of the amyloid-β peptide derived from cleavage of the amyloid precursor protein (APP). Mutations in APP lead to the development of Familial Alzheimer''s Disease (FAD), however, the normal function of this protein has proven elusive. The organism Caenorhabditis elegans is an attractive model as the amyloid precursor-like protein (APL-1) is the single ortholog of APP, and loss of apl-1 leads to a severe molting defect and early larval lethality.

Methodology/Principal Findings

We report here that lethality and molting can be rescued by full length APL-1, C-terminal mutations as well as a C-terminal truncation, suggesting that the extracellular region of the protein is essential for viability. RNAi knock-down of apl-1 followed by drug testing on the acetylcholinesterase inhibitor aldicarb showed that loss of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. The aldicarb hypersensitivity can be rescued by full length APL-1 in a dose dependent fashion. At the cellular level, kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are positive regulators of APL-1 expression in the neurons. Knock-down of the small GTPase rab-5 also leads to a dramatic decrease in the amount of apl-1 expression in neurons, suggesting that trafficking from the plasma membrane to the early endosome is important for apl-1 function. Loss of function of a different small GTPase, UNC-108, on the contrary, leads to the retention of APL-1 in the cell body.

Conclusions/Significance

Our results reveal novel insights into the intracellular trafficking of APL-1 and we report a functional role for APL-1 in synaptic transmission.     

Structural Characterization of the E2 Domain of APL-1, a Caenorhabditis elegans Homolog of Human Amyloid Precursor Protein, and Its Heparin Binding Site

The amyloid β-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.     

Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis

Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.     

What the evolution of the amyloid protein precursor supergene family tells us about its function

The Alzheimer's disease amyloid protein precursor (APP) gene is part of a multi-gene super-family from which sixteen homologous amyloid precursor-like proteins (APLP) and APP species homologues have been isolated and characterised. Comparison of exon structure (including the uncharacterised APL-1 gene), construction of phylogenetic trees, and analysis of the protein sequence alignment of known homologues of the APP super-family were performed to reconstruct the evolution of the family and to assess the functional significance of conserved protein sequences between homologues. This analysis supports an adhesion function for all members of the APP super family, with specificity determined by those sequences which are not conserved between APLP lineages, and provides evidence for an increasingly complex APP superfamily during evolution. The analysis also suggests that Drosophila APPL and Caenorhabditis elegans APL-1 may be a fourth APLP lineage indicating that these proteins, while not functional homologues of human APP, are similarly likely to regulate cell adhesion. Furthermore, the betaA4 sequence is highly conserved only in APP orthologues, strongly suggesting this sequence is of significant functional importance in this lineage.     

LET-60 RAS modulates effects of insulin/IGF-1 signaling on development and aging in Caenorhabditis elegans

The DAF-2 insulin/insulin-like growth factor 1 (IGF-1) receptor signals via a phosphatidylinositol 3-kinase (PI3K) pathway to control dauer larva formation and adult longevity in Caenorhabditis elegans. Yet epistasis analysis suggests signal bifurcation downstream of DAF-2. We have used epistasis analysis to test whether the Ras pathway (which plays a role in signaling from mammalian insulin receptors) acts downstream of DAF-2. We find that an activated Ras mutation, let-60(n1046gf), weakly suppresses constitutive dauer diapause in daf-2 and age-1 (PI3K) mutants. Moreover, increased Ras pathway signaling partially suppresses the daf-2 mutant feeding defect, while reduced Ras pathway signaling enhances it. By contrast, activated Ras extends the longevity induced by mutation of daf-2, while reduced Ras pathway signaling partially suppresses it. Thus, Ras pathway signaling appears to act with insulin/IGF-1 signaling during larval development, but against it during aging.     

acn-1, a C. elegans homologue of ACE,genetically interacts with the let-7 microRNA and other heterochronic genes

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors – lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.     

The secreted Alzheimer-related amyloid precursor protein fragment has an essential role in C. elegans

Mutations in the gene encoding the amyloid precursor protein (APP) or the enzymes that process APP are correlated with familial Alzheimer disease. Alzheimer disease is also associated with insulin resistance (type 2 diabetes). In our recently published study,¹ Ewald CY, Raps DA, Li C. APL-1, the Alzheimer’s Amyloid precursor protein in Caenorhabditis elegans, modulates multiple metabolic pathways throughout development. Genetics 2012; 191:493 - 507; http://dx.doi.org/10.1534/genetics.112.138768; PMID: 22466039 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] we obtained genetic evidence that the extracellular fragment of APL-1, the C. elegans ortholog of human APP, may act as a signaling molecule to modulate insulin and nuclear hormone pathways in C. elegans development. In addition, independent of insulin and nuclear hormone signaling, high levels of the extracellular fragment of APL-1 (sAPL-1) leads to a temperature-sensitive embryonic lethality, which is dependent on activity of a predicted receptor protein tyrosine phosphatase (MOA-1/R155.2). Furthermore, this embryonic lethality is enhanced by knockdown of a predicted prion-like protein (pqn-29). The precise molecular mechanisms underlying these processes remain to be determined. Here, we present hypothetical models as to how sAPL-1 signaling influences metabolic and developmental pathways. Together, with previous findings in mammals that the extracellular domain of mammalian APP (sAPP) binds to a death-receptor,² Nikolaev A, McLaughlin T, O’Leary DD, Tessier-Lavigne M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 2009; 457:981 - 9; http://dx.doi.org/10.1038/nature07767; PMID: 19225519 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] our findings support the model that sAPP signaling affects critical biological processes.     

Transposon insertion mutagenesis of a genetic region encoding serum resistance in an 80 kb plasmid of Salmonella dublin

Using transposon insertion mutagenesis with Tn1 or Tn5, we obtained Salmonella dublin mutant strains that showed either diminished serum resistance (five mutants) or diminished mouse lethality (two mutants). Detailed restriction cleavage analysis to determine the single sites of transposon insertion in an 80 kb plasmid (pTE800) indicated that a region for serum resistance was located within a 3.0 kb region of the SalI cleavage fragment 5 and the HindIII fragment 2, while the region for mouse lethality was within a 6.0 kb region of the SalI fragment 2 and the HindIII fragment 1. When the Tn1-containing SalI fragment 5 was reconverted, by homologous recombination, to the original SalI fragment 5 (9.6 kb), serum resistance was recovered to the same level as that of a parent strain 52401. Moreover, the change in the serum resistance correlated with changes in the neutral sugar composition of the LPS. The mutation in the plasmid in strain TE4-55 that gave diminished mouse lethality was also reversed by recombination with the cloned SalI fragment 2 (15.0 kb), with concomitant recovery of mouse lethality. These results indicate that the genetic region for serum resistance is different from that for mouse lethality, and that the gene for serum resistance is closely involved with the expression of the neutral sugar composition of the LPS of S. dublin.     

Caenorhabditis elegans SDF-9 enhances insulin/insulin-like signaling through interaction with DAF-2

SDF-9 is a modulator of Caenorhabditis elegans insulin/IGF-1 signaling that may interact directly with the DAF-2 receptor. SDF-9 is a tyrosine phosphatase-like protein that, when mutated, enhances many partial loss-of-function mutants in the dauer pathway except for the temperature-sensitive mutant daf-2(m41). We propose that SDF-9 stabilizes the active phosphorylated state of DAF-2 or acts as an adaptor protein to enhance insulin-like signaling.     

Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25

In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.