APL-1, a Caenorhabditis elegans protein related to the human β-amyloid precursor protein, is essential for viability

Abstract

Dominant mutations in the amyloid precursor protein (APP) gene are associated with rare cases of familial Alzheimer's disease; however, the normal functions of APP and related proteins remain unclear. The nematode Caenorhabditis elegans has a single APP-related gene, apl-1, that is expressed in multiple tissues. Loss of apl-1 disrupts several developmental processes, including molting and morphogenesis, and results in larval lethality. The apl-1 lethality can be rescued by neuronal expression of the extracellular domain of APL-1. These data highlight the importance of the extracellular domain of an APP family member and suggest that APL-1 acts noncell-autonomously during development. Overexpression of APL-1 also causes several defects, including a high level of larval lethality. Decreased activity of sel-12, a C. elegans homologue of the human γ-secretase component presenilin 1, partially rescues the lethality associated with APL-1 overexpression, suggesting that SEL-12 activity regulates APL-1 activity either directly or indirectly.

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder that is characterized pathologically by the accumulation of dense plaques in the brains of AD patients. The main component of these plaques is the β-amyloid peptide (1, 2), which is a cleavage product of the amyloid precursor protein (APP; ref. 3). Autosomal dominant mutations in APP have been correlated with a small number of early-onset AD cases (see Alzheimer's Disease Mutation Database at www.molgen.ua.ac.be/ADMutations). Although APP has been implicated in several processes in vitro, such as neurite outgrowth, cell adhesion, and cell survival (for review see ref. 4), the in vivo functions of APP remain unclear.

Determining the in vivo functions of APP in mammals is complicated by the presence of two APP-related genes, APLP1 and APLP2 (for review see ref. 5). APP and APP-related proteins share two conserved domains in the extracellular region (E1 and E2) and one in the cytoplasmic domain, but the APP-related proteins do not contain the β-amyloid peptide (5). Mice in which APP, APLP1, or APLP2 is inactivated are viable and have minor behavioral and growth deficits (6–8). However, inactivation of APLP2 and either APP or APLP1 results in early postnatal lethality (6, 8), indicating that the APP family is essential for viability. The brains of double knockout animals exhibit no obvious morphological defects (6, 8). By contrast, animals in which the entire APP gene family is inactivated show cortical dysplasia and type 2 lissencephaly, indicating that the APP gene family is necessary for neurodevelopment and adhesion (9).

Although no APP gene has been identified in Drosophila melanogaster or Caenorhabditis elegans, each organism contains a single APP-related gene (10, 11). Inactivation of the Drosophila APP-related gene, Appl, causes abnormal synaptic differentiation (12), axonal transport (13, 14), and phototactic behavior (15), the latter of which can be partially rescued with a human APP transgene (15). Expression of human APP in Drosophila wing imaginal discs results in a blistered wing phenotype, showing that overexpression of APP can disrupt cell adhesion in the transgenic animals (16).

In this article, we examine the role of apl-1 in C. elegans. Zambrano et al. (17) have reported mild pharyngeal defects when apl-1 activity is decreased by dsRNA-mediated interference by feeding. We genetically inactivated apl-1 and found that, like the mammalian APP gene family, apl-1 has an essential role in C. elegans. In particular, APL-1 is necessary for proper molting and morphogenesis. Furthermore, expression of the extracellular domain of APL-1 in neurons is sufficient to rescue the apl-1 lethality. These data highlight the significance of the extracellular domain of APL-1 and perhaps other APP-related proteins.

Results and Discussion

apl-1 Is Expressed in Multiple Cell Types.

apl-1 (C42D8.8) maps to the X chromosome and contains 12 exons (Fig. 1A). Like mammalian APP (3), APL-1 undergoes glycosylation and cleavage to release a large extracellular domain (sAPL-1; Fig. 1D). To determine the expression pattern of apl-1, we generated animals carrying transcriptional and translational GFP reporter transgenes. All transgenic lines exhibited comparable expression patterns. Similar to the widespread expression of mammalian APP (for review see ref. 18), apl-1 expression was detected in >50 neuronal, muscle, hypodermal, and supporting cells in adults [Fig. 2 and supporting information (SI) Fig. 4]. The larval expression pattern was similar to the adult pattern with a few exceptions; for instance, apl-1 is expressed in more ventral cord motor neurons in the first larval stage (L1) animals than in other larval stages or adults (data not shown).

Fig. 1.

Molecular characterization of apl-1 alleles. (A) Schematic of the apl-1 genomic locus. The gray boxes represent the regions encoding the extracellular domain of APL-1, the striped box indicates the transmembrane (TMD) and cytoplasmic domains, and the open box indicates the 3′ UTR. apl-1 is trans-spliced, so the start site of transcription is unknown. Locations of the Tc1 insertion (inverted triangle), apl-1 point mutations, and yn5 and yn10 deletions are indicated (see SI Text for exact lesions). AMB, OCH, and OPA indicate amber, ochre, and opal stop codons, respectively. (B) Schematic of proteins encoded by constructs tested for rescue of the apl-1 lethality. The signal sequence (circles), extracellular E1 (amino acids 1–209) and E2 (amino acids 236–423) domains, TMD (gray box), cytoplasmic domain (C), and putative G_oα binding site (striped boxes) are shown. Chimeric proteins with GFP are indicated; APL-1::GFP lines carry an apl-1 genomic fragment or cDNA fused to GFP. The number of lines that rescued the given apl-1 mutation relative to the total number of independent transgenic lines is shown. ND, not determined. (C) yn10, yn23, and yn32 are likely null alleles. To maintain apl-1 homozygotes, all lines carried a rescue construct (listed in brackets below but not shown) except yn5. All Western blots were probed with an antibody against the entire APL-1 extracellular domain (APL-1EXT). Extracts from WT animals contained full-length APL-1 (arrowhead in top blot). sAPL-1, the cleaved extracellular domain of full-length APL-1, is not visible in this blot. Extracts from lon-2 yn10;[APL-1EXT] (labeled yn10) only contain APL-1EXT (dot in top blot) from the rescue construct. No full-length APL-1 (arrowhead in top blot) or protein corresponding to in vitro-translated yn10 protein (yn10IV) (star in second blot) was detected. Homozygous yn5 deletion mutants are viable and produce only APL-1EXT (dot in top blot). yn23 dpy-8;[APL-1::GFP] animals (labeled yn23) only contained APL-1::GFP (double arrowhead) and its cleavage product, sAPL-1 (open dot) from the rescue construct (third blot). No full-length APL-1 (arrowhead in third blot) or protein corresponding to in vitro-translated yn23 protein (star in fourth blot) was detected. yn5IV is shown as a size control. yn32 is a missense mutation predicted to allow full-length APL-1 production. However, extracts from lon-2 yn32;[APL-1EXT] (labeled yn32) only contained APL-1EXT from the rescue construct (dot in bottom blot). No full-length APL-1 (arrowhead in bottom blot) or cleaved sAPL-1 (open dot in bottom blot) was detected. Extracts from yn5 animals were run as a size control. Note that APL-1EXT is slightly larger than sAPL-1. Molecular mass markers and estimated weights are in kDa. (D) Expression of high levels of APL-1 in yn5 and integrated trangenic apl-1 strains in a WT or decreased sel-12 γ-secretase background. APL-1 expression driven by a pan-neuronal promoter is indicated by nAPL-1. Note that the amount of total protein loaded on the Western blot (indicated at the bottom) is varied up to 20-fold to allow visualization of WT protein. Extracts from WT animals contain full-length and glycosylated APL-1 (arrowhead; 105–110 kDa), a cleaved extracellular fragment sAPL-1 (open dot; ≈85 kDa), and high molecular mass forms that are presumably dimers (asterisk; ≈200–220 kDa). yn5 extracts contain only APL-1EXT (dot; ≈90 kDa), which is slightly larger than sAPL-1 because it does not appear to be further cleaved by secretases. Transgenic apl-1 overexpression lines (ynIs79 APL-1::GFP, ynIs86 APL-1, ynIs13 nAPL-1, and ynIs12 nAPL-1) are in a WT or sel-12 γ-secretase mutant background. ynIs79 APL-1::GFP animals contain proteins corresponding to sAPL-1 (open dot), APL-1::GFP (double arrowheads; ≈135 kDa), and high molecular mass forms (double asterisk; ≈260 kDa) that are presumably dimers of APL-1::GFP; the high levels of presumptive dimers may be caused by high levels of APL-1 or because the GFP tag promotes dimerization. The endogenous WT protein is not detectable at this concentration of total protein in ynIs79 APL-1::GFP animals. Other transgenic animals contain full-length and glycosylated APL-1 (arrowhead), sAPL-1 (open dot), and low levels of presumptive dimers of APL-1 (asterisk). WT and yn5 in vitro-translated proteins are shown as size controls; smaller IV proteins are presumably caused by initiation from inappropriate downstream methionine codons. Molecular mass markers are in kDa. Quantification of APL-1 levels is in SI Table 2. See SI Text for details about methods.

Fig. 2.

APL-1 is expressed in multiple cell types. (A) Head region of an adult animal carrying a GFP transcriptional reporter construct under the control of an apl-1 promoter shown in a lateral view. apl-1 is expressed in a variety of cell types, including neurons (indicated by their three letter names; where the identity of the neurons is between two cells, both possibilities are indicated), muscle cells, and supporting cells. Many processes in the nerve ring, the major neuropil region of the animal, and various nerve bundles (arrows) also express apl-1. Gut granules within intestinal cells (arrowheads) often show nonspecific fluorescence. MNs, motor neurons. (B) In ynIs79 APL-1::GFP animals, GFP expression is faint and punctate, suggesting that APL-1::GFP is located in vesicular compartments of the cell. The cells that exhibit apl-1 expression, however, are the same as those in A. (Scale bars: A, 25 μm; B, 10 μm.)

apl-1 Is Essential for Viability.

To determine the function of apl-1, we isolated a strain (pk53 apl-1:Tc1) containing a Tc1 transposon insertion in the apl-1 gene (Fig. 1A) and screened for imprecise transposon excisions that deleted parts of the apl-1 coding region. We isolated two deletion mutants: yn5 (see below) and yn10 (Fig. 1A and SI Text. Because yn10 mutants produced no detectable APL-1 protein (Fig. 1C), we concluded that yn10 is a null allele. To isolate additional apl-1 alleles, we performed F1 noncomplementation screens and isolated five more alleles: yn23, yn29, yn30, yn31, and yn32. All of the isolated alleles correspond to point mutations within the coding region of the extracellular domain and cause phenotypes similar to yn10 (Fig. 1A and SI Text).

apl-1(yn10) and the isolated point mutations cause a recessive larval lethal phenotype. To determine when apl-1 is required, we observed the development of yn10 animals. Most mutants were morphologically WT at hatching. Shortly after hatching, however, the mutants (35/41 animals) began to accumulate variously sized vacuoles (Fig. 3C, D, and F) that appeared to be within the large syncytial hypodermal cells. A few mutants (4/30 animals) arrested or died during L1 because of severe morphological defects (Fig. 3C). A small number of L1 animals (3/30 animals) had large gaps between organs, perhaps because of an adhesion or osmoregulatory defect (Fig. 3E). As monitored by a neural-specific GFP marker, the nervous system of L1 mutants appeared normal and axon bundles were correctly positioned (data not shown). No other phenotype was seen until the first to second larval stage (L2) transition when all mutant animals displayed a molting defect. During each larval stage transition, WT animals synthesize a new cuticle and shed their old cuticle. The mutants synthesized a new L2 cuticle but failed to shed their old L1 cuticle, thereby becoming trapped within the old cuticle and dying (Fig. 3F). Hence, inactivation of apl-1 disrupts molting and morphogenesis.

Fig. 3.

Either loss or overexpression of apl-1 causes morphological defects. (A and B) Head (A) and gonadal (B) regions of a WT first larval stage (L1) hermaphrodite animal. (C–F) Homozygous yn10 hermaphrodites in a sem-4;lon-2 background. The sem-4 mutation, which causes adult hermaphrodites to retain their eggs, was used to aid in the observation of embryos. Similar phenotypes were seen in a non-sem-4 background. (C) An L1 animal in which posterior morphogenesis was disrupted. The midregion of the animal is shown. Posterior organs did not develop and the midregion ends abruptly (arrows); the cuticle, however, extended posteriorly. Vacuoles (double arrows) can be seen throughout the animal. (D) A late L1/early L2 animal that showed large vacuoles (double arrows) is shown. (E) A late L1/early L2 animal in which a large gap between the gonad and overlying intestine was seen (arrow). (F) A late L1/early L2 animal that had not shed its old cuticle (arrowheads), which remained attached at its mouth (arrow). The animal also had a large vacuole (double arrow). (G and H) An L1 apl-1 overexpression animal (ynIs79 APL-1::GFP) had a translucent appearance. Cells and organs appeared in stark relief, particularly in the midregion of the animal (H). Arrows point to muscle nuclei. Anterior is to the left. All micrographs were taken on live animals; except for WT, none of the animals were anesthetized. ph, first bulb of pharynx, nr, nerve ring region, g, gonad, int, intestine. (Scale bars: 10 μm.)

To confirm that these phenotypes were caused by loss of apl-1, we transformed apl-1 heterozygotes with an 8.45-kb apl-1 genomic fragment and recovered transgenic homozgyous apl-1 progeny that were rescued for all apl-1 mutant phenotypes (SI Fig. 5). By contrast, the same genomic fragment containing point mutations in the coding region failed to rescue the apl-1 lethality (SI Fig. 5). Expression of an APL-1::GFP fusion protein using apl-1 regulatory sequences also rescued the lethality (Fig. 1B). Together, these data indicate that the observed phenotypes result specifically from loss of apl-1 function. The early lethality in apl-1 mutants hampers an examination of apl-1 function after the first molt, so the full extent of APL-1 activity is unknown.

To determine whether there is a maternal apl-1 contribution, we examined homozygous yn10 mothers that carried extrachromosomal rescue arrays, which can be lost at some frequency during cell division, thereby creating mosaic animals in which some cells contain the transgene, whereas other cells do not. Mosaic mothers in which the rescue arrays were lost from the germ line produce progeny that lack maternal and zygotic apl-1 activity. All such mosaic mothers (n = 21) produced only dead L1 or L1/L2 animals, which were similar in phenotype to apl-1 homozygotes produced from apl-1 heterozygotes. We conclude, therefore, that there is little or no maternal apl-1 contribution.

To evaluate apl-1 transcript and protein levels in the mutants, we performed RT-PCR and Western blot analysis on yn10, yn23, and yn32 animals, which are homozygous for deletion, nonsense, and missense mutations, respectively; the mutant animals all carried rescue transgenes, which produce WT products that could be differentiated from the endogenous mutant apl-1 transcripts and proteins. apl-1 transcripts are detected in WT eggs, larvae, and adults (19). Similarly, mutant transcripts were isolated from all three mutants (data not shown). However, no endogenous mutant APL-1 protein was detected in any of the three strains (Fig. 1C), indicating that these three mutations are likely null alleles. The yn32 mutation causes a glutamic acid to lysine substitution at residue 372 in the E2 domain. In human APP the corresponding residue is not predicted to be important for the structural stability of E2 (20); however, this residue is proposed to be part of an α-helix that is necessary for APP cleavage (21). Animals carrying an apl-1(yn32)::GFP transgene express GFP faintly (data not shown), suggesting that yn32 animals die from insufficient levels of APL-1::yn32 protein because it is unstable, degraded quickly, or not cleaved to produce sAPL-1.

To address whether human APP is a functional homologue of apl-1, we determined whether human APP expressed using apl-1 regulatory sequences could rescue the apl-1 mutants. This construct was unable to rescue yn10 (0/20 rescued lines). We have not determined whether other human APP, APLP1, APLP2, or Drosophila Appl constructs are able to rescue apl-1 mutants.

The apl-1-Induced Lethality Is Not Caused by Activation of Cell Death Pathways.

Mammalian APP has been proposed to regulate apoptosis (22), yet APP can also be cleaved by different caspases (23–25). We, therefore, examined whether the apl-1 lethality is caused by activation of an apoptotic or necrotic cell death pathway. In C. elegans ced-3 encodes a caspase that is essential for execution of apoptosis (26) and crt-1 encodes calreticulin, which is essential for execution of necrotic cell deaths (27). Neither loss of ced-3 caspase nor loss of crt-1 calreticulin activity rescued the yn10 lethality, indicating that the apl-1 lethality is not caused by ectopic activation of either cell death pathway.

Expression of the Extracellular Domain of APL-1 Is Sufficient for Viability.

Different domains of human APP have been shown to interact with a number of proteins in vitro (for reviews see refs. 28 and 29). For example, the APP extracellular domain interacts with Notch (30), low-density lipoprotein receptor-related protein (31), and components of the extracellular matrix (4, 28, 29). Similarly, defined regions of the APP cytoplasmic domain interact with several proteins, such as G_oα, Disabled, and kinesin-1 (14, 28, 29); in addition, the APP cyptoplasmic tail, when complexed with Fe65, has been proposed to enter the nucleus to affect transcription (32). The biological significance of these interactions, however, remains unclear.

To determine which domains of APL-1 are necessary for function in vivo, we tested whether apl-1 transgenes that were deleted for specific domains were sufficient for rescue of the apl-1 lethality. As much research has focused on interactions of the cytoplasmic domain of mammalian APP, we first investigated whether cytoplasmic domains within APL-1 were needed for viability. Deletion of the putative G_oα binding site (11) did not affect transgene rescue of the apl-1 lethality (Fig. 1B). Strikingly, an APL-1 protein lacking the transmembrane and cytoplasmic domains also gave complete rescue (Fig. 1B), indicating that the activities necessary for rescue are contained within the extracellular domain of APL-1. Consistent with this result, apl-1(yn5) deletion mutants are homozygous viable and produce only the extracellular domain of APL-1 (APL-1EXT) (Fig. 1D), which is slightly larger than the WT cleaved APL-1 (sAPL-1). These data argue that the extracellular domain is critical for APL-1 rescuing activity and that the lethality is not caused by the lack of either protein interactions with the cytoplasmic domain of APL-1 or apl-1-dependent transcription.

To determine which regions of the APL-1 extracellular domain are necessary for rescue, we deleted sequences encoding the E1 domain (ΔE1), the E2 domain (ΔE2), or the E1 through most of the E2 domains (ΔE1-E2) and tested the modified constructs for rescuing activity. Both APL-1ΔE1 and APL-1ΔE2 were each able to rescue apl-1 mutants, whereas APL-1ΔE1-E2 did not (Fig. 1B). Thus, despite structural dissimilarities, either the E1 or E2 domain by itself is sufficient for viability and the two domains function independently and redundantly. The E1 and E2 domains of mammalian APP have been shown to bind heparin, collagen, and laminin, presumably to mediate cell–cell or cell–substratum adhesion or increase neurite outgrowth (28, 29); APL-1 may similarly bind the extracellular matrix to mediate equivalent functions.

apl-1 Expression in Neurons Is Sufficient for Viability.

To determine the site of action of apl-1, we expressed full-length APL-1::GFP by using different tissue-specific promoters. Expression of APL-1 in pharyngeal muscle, body wall muscles, or hypodermal cells failed to rescue the apl-1(yn10) lethality (0/4, 0/6, and 0/3 rescued lines, respectively). By contrast, APL-1 expression driven by either the pan-neuronal snb-1 (3/3 yn10 and 1/1 yn23 rescued lines) or rab-3 (4/4 rescued yn10 lines) promoters (nAPL-1; refs. 33 and 34) was sufficient to rescue the apl-1 lethality. Furthermore, expression of APL-1EXT in neurons with the snb-1 promoter also rescued the apl-1 lethality (6/6 yn10 and 1/1 yn32 rescued lines), suggesting that cleavage of full-length APL-1 to release sAPL-1 from neurons is sufficient for proper molting and morphogenesis. The failure to rescue by hypodermal expression of APL-1 was puzzling, because sAPL-1 is presumably affecting the hypodermal cells to influence molting. However, hypodermal cells might lack the appropriate proteases to cleave APL-1 to generate sAPL-1.

Overexpression of APL-1 Causes Defects in Brood Size, Movement, and Viability.

To determine whether APL-1 overexpression caused defects, we analyzed several transgenic lines in which the apl-1 rescuing transgenes were integrated into the genome and crossed into a WT background. These strains include ynIs86, which contains integrated copies of APL-1; ynIs79, which contains integrated copies of APL-1::GFP; and ynIs12 and ynIs13, which contain integrated copies of nAPL-1. By Western blot analysis, APL-1 or APL-1::GFP (which will be referred collectively as APL-1 henceforth) levels were at least 15-fold higher in the transgenic overexpression lines compared with WT (Fig. 1D and SI Table 2). A similar increase in the level of APL-1EXT was also observed in apl-1(yn5) mutants (Fig. 1D and SI Table 2). This increased APL-1EXT level in yn5 mutants may be caused by constitutive release of APL-1EXT or increased stability and/or resistance to the degradation of APL-1EXT.

Transgenic strains overexpressing APL-1 had defects in brood size, movement, and viability; the severity of these defects was strongly correlated with the level of APL-1 overexpression (Table 1, SI Table 2, and Fig. 1D). Wild-type animals generally lay between 250 to 300 eggs (ref. 35 and Table 1). Animals overexpressing APL-1 laid significantly fewer eggs than WT (Table 1). Because apl-1(yn5) APL-1EXT animals also showed a decreased number of progeny (Table 1), we hypothesize that brood size is decreased by elevated levels of cleaved APL-1. The increased level of extracellular APL-1 may interfere with cell–cell interactions, thereby disrupting morphogenesis and/or gonadal development.

Table 1.

Overexpression of apl-1 causes decreased brood size and sluggishness

Genotype	Brood size			Movement
	No. of eggs	No. of adult progeny	% survival	No. of thrashes per min	No. of head bends per min
WT	250.6 ± 5.3 (n = 46)	261.6 ± 5.0 (n = 46)	100	86.4 ± 2.7 (n = 60)	15.5 ± 0.7 (n = 60)
yn5APL-1EXT	187.9 ± 4.9* (n = 33)	192.2 ± 5.0* (n = 33)	100	77.5 ± 5.0 (n = 30)	14.0 ± 0.9 (n = 40) Transgenic apl-1 overexpression lines†
Transgenic apl-1 overexpression lines†
ynIs86APL-1	203.8 ± 8.7* (n = 43)	192.6 ± 7.0* (n = 43)	94.5	56.0 ± 3.0* (n = 30)	11.8 ± 0.8 (n = 44)
ynIs79 APL-1::GFP	217.6 ± 7.9 (n = 31)	64.6 ± 5.1* (n = 31)	29.7	32.9 ± 3.3* (n = 31)	6.9 ± 0.6* (n = 39)
ynIs13nAPL-1	228.3 ± 4.8 (n = 43)	229.7 ± 5.0* (n = 43)	100	52.0 ± 3.2* (n = 30)	11.8 ± 0.9 (n = 40)
ynIs13; ynIs86	209.8 ± 4.4* (n = 50)	197.3 ± 4.2 (n = 50)	94.0	16.1 ± 1.4* (n = 31)	5.4 ± 0.6* (n = 40) Decreased sel-12 γ-secretase activity in apl-1 overexpression lines
Decreased sel-12 b-secretase activity in apl-1 overexpression lines
sel-12 γ-secretase	103.2 ± 6.2* (n = 48)	112.8 ± 7.3* (n = 48)	100	84.2 ± 1.9 (n = 20)	18.9 ± 1.9 (n = 19)
sel-12 yn5	86.3 ± 10.4* (n = 43)	90.9 ± 0.9.9* (n = 43)	100	73.2 ± 3.3*‡ (n = 40)	12.6 ± 0.9* (n = 40)
sel-12;ynIs79	103.4 ± 8.5* (n = 56)	72.8 ± 6.1* (n = 56)	70.4	11.8 ± 1.1*‡ (n = 20)	8.4 ± 1.0*‡ (n = 19)
sel-12 ynIs86	54.7 ± 7.0*‡ (n = 19)	59.1 ± 7.8*‡ (n = 19)	100	35.7 ± 5.3*‡ (n = 20)	11.4 ± 0.9‡ (n = 20)

Values represent the means ± SEM. n, the total number of animals analyzed. nAPL-1 indicates neuronal expression driven by the snb-1 promoter. Because eggs and newly hatched L1 animals are harder to visualize than adults, the number of adult progeny is sometimes greater than the number of eggs laid; these cases are indicated as 100%.

*Significantly different from WT (P < 0.01; one-way ANOVA; Newman–Keuls post hoc test).

^†ynIs86, ynIs79, and ynIs13 are transgenic lines that contain integrated arrays of APL-1, APL-1::GFP, and nAPL-1, respectively.

^‡Significantly different from sel-12 (P < 0.01; one-way ANOVA; Newman–Keuls post hoc test).

Transgenic animals overexpressing APL-1 also had subtle movement defects, which we quantified in two motor assays. When placed into physiological buffer, WT animals begin to swim, and the number of body flexures or thrashes per minute can be quantified (Table 1). All apl-1 transgenic overexpression strains showed a significantly reduced thrashing rate (Table 1). Second, on a solid surface WT animals move in a sinusoidal waveform, which can be quantified by counting the number of head bends per min. All transgenic overexpression strains had a decreased head bend rate compared with WT (Table 1). Furthermore, a transgenic line that was homozygous for two integrated transgenes produced higher levels of APL-1 (Fig. 1D and SI Table 2) and was more sluggish than either parental line alone (Table 1). Increasing levels of APL-1, therefore, inhibit movement, perhaps by interfering with motor neuron functions.

Several APL-1 overexpressing strains, particularly apl-1(yn5) APL-1EXT animals, developed more slowly than WT (SI Table 3). Wild-type animals lay eggs that hatch and develop into adults in ≈3 days at 20°C (SI Table 3). By contrast, after 3 days, the majority of apl-1(yn5) APL-1EXT progeny were still in a late larval stage (SI Table 3). Transgenic overexpression animals also showed varying degrees of developmental delay (SI Table 3), suggesting that higher levels of APL-1 and/or APL-1EXT interfere with the normal developmental progression of the animal.

The ynIs79 APL-1::GFP overexpression line exhibited the most severe phenotypes and expressed the highest levels of APL-1 (≈180-fold higher than WT; Fig. 1D and SI Table 2). Interestingly, although ynIs79 APL-1::GFP overexpression animals laid a similar number of eggs compared with other transgenic overexpression lines, ≈70% of the animals died during L1 (Table 1), and this lethality rate was unchanged in a ced-3 caspase loss-of-function background (data not shown). No such lethality was associated with apl-1(yn5) or other transgenic overexpression lines, except ynIs86 APL-1 animals, which showed a low lethality rate (Table 1). ynIs79 APL-1::GFP overexpression animals appear morphologically WT at hatching. At variable times during L1, ynIs79 APL-1::GFP overexpression animals become translucent and large gaps are present between organs (Fig. 3 G and H). Whereas in WT animals muscle bundles appear contiguous with intestinal and gonadal cells, in ynIs79 APL-1::GFP overexpression animals muscle bundles are clearly separated from intestinal cells and the gonad when visualized by Nomarski optics (Fig. 3H). When visualized by fluorescently tagged phalloidin, the fibers within the muscle bundles of ynIs79 APL-1::GFP overexpression animals were difficult to visualize, as though the muscle bundles were condensed (data not shown). These phenotypes are consistent with disruptions in cell adhesion, whereby elevated expression of APL-1 interferes with the normal adhesion contacts between cells, or in osmoregulation.

Loss of sel-12 Presenilin Partially Rescues the Lethality Caused by apl-1 Overexpression.

Presenilin 1 (PS1) has been proposed to be part of the γ-secretase complex that cleaves substrates such as human APP and the LIN-12/Notch receptor (for review see ref. 36); in addition, PS1 may regulate the level, amount of cleavage, and/or trafficking of APP (37, 38). One C. elegans homologue of PS1 is sel-12, which was identified as a suppressor of a lin-12 gain-of-function mutation (39). If SEL-12 similarly regulates levels, processing, and/or trafficking of APL-1, the amount of lethality seen in ynIs79 APL-1::GFP overexpression animals might be altered by loss of sel-12 activity. Reduced sel-12 activity causes an egg-laying defect that decreases the number of eggs (39), but does not affect the viability of the progeny (Table 1). The lethality of ynIs79 APL-1::GFP overexpression was partially rescued (30% compared with 70% lethality) by reduced sel-12 activity (Table 1). However, all forms of APL-1, including cleaved, full-length, and high-molecular-weight forms of APL-1, were present in ynIs79 APL-1::GFP; sel-12 animals in roughly the same proportions but at slightly lower levels than in ynIs79 APL-1::GFP animals alone (Fig. 1D and SI Table 2). Thus, sel-12 does not appear to affect the α-secretase cleavage of APL-1. We propose that SEL-12, like mammalian PS1, either directly or indirectly regulates levels and/or trafficking of APL-1 and a change in this regulation affects the viability of the animals.

Concluding Remarks

Our results indicate that APL-1 is an essential, multifunctional protein involved in molting, reproduction, locomotion, and morphogenesis. Our finding that release of APL-1EXT from neurons is sufficient for viability highlights the importance of the extracellular domain of an APP-related protein and suggests that the source of the cleaved APL-1 fragment sAPL-1 and its spatial distribution are critical. We suggest that the extracellular domains of mammalian APP and related proteins may also have critical functions in the nervous system.

Materials and Methods

Strains.

C. elegans strains were grown at 20°C and maintained according to Brenner (40). The mutations used are as described in Wormbase (www.wormbase.org) and include: LGI, sem-4(n1378); LGV, crt-1(ok948); and LGX, lon-2(e678), dpy-8(e130), lin-15(n765), sel-12(ar131). Integrated transgenic lines were: LGIII, ynIs12 (nAPL-1; P_snb-1::apl-1); LGV, ynIs79 APL-1::GFP, ynIs13 (nAPL-1; P_snb-1::apl-1); and LGX, ynIs86 APL-1. Nonintegrated transgenic lines used were: ynEx106 APL-1EXT, ynEx106A APL-1EXT, ynEx165 (P_snb-1::APL-1EXT), and ynEx166 (P_snb-1::APL-1EXT). CL1093 (P_apl-1::GFP) was a kind gift of Chris Link (University of Colorado, Boulder, CO).

Identification of apl-1 Alleles.

All isolated mutants were backcrossed, and molecular lesions were determined by DNA sequencing. pk53 (apl-1::Tc1) was isolated by screening a mutant bank of transposon-insertion strains (41). The yn5 and yn10 mutations were isolated in a PCR-based screen of 2,340 pk53 (apl-1::Tc1) populations (41). F₁ noncomplementation screens were used to isolate yn23, yn29, yn30, yn31, and yn32 from a total of 3,105 mutagenized haploid genomes (see SI Text).

Construction and Microinjection of Test Plasmids.

Several derivatives of the cosmid C42D8, including two with point mutations in the apl-1 coding region, were tested for rescue (SI Fig. 5 and SI Text). Constructs containing different deletions in apl-1 were generated by PCR and sequenced to verify the deletions (see SI Text). Heterologous promoters used to drive apl-1 cDNA expression were: the pan-neuronal promoters synaptobrevin (snb-1) (33) and rab-3 (34); the pharyngeal and body wall muscle promoters myo-2 and myo-3, respectively (42); and the hypodermal cell col-10 promoter (43). Constructs were microinjected at 50–150 ng/μl; dominant markers (50 ng/μl) coinjected to identify transgenic animals were pRF4 rol-6 (44), pTG96 sur-5::GFP (45), or pJM24 lin-15 (46). GFP-tagged constructs were generally not coinjected with marker plasmids. The transgenic arrays were not integrated into the genome unless otherwise noted.

Determination of apl-1 Rescue.

Test constructs were coinjected into heterozygous lon-2 apl-1(yn10)/dpy-8, apl-1(yn23) dpy-8/lon-2, or lon-2 apl-1(yn32)/dpy-8 animals with the marker plasmids pRF4 rol-6 (44) or pTG96 sur-5:GFP (45) unless the construct was GFP-tagged. The progeny of the injected animals were tested for rescue as follows. The presence of adult progeny with the phenotype caused by the genetic marker in cis to apl-1 (i.e., Lon or Dpy) suggested rescue by the injected transgene. These transgenic progeny were individually plated and allowed to self-fertilize. Confirmation that the strain was homozygous for the apl-1 allele was done by PCR, PCR followed by restriction digests, and/or DNA sequence analysis. The presence of GFP or the coinjection marker (i.e., rol-6 or sur-5::GFP) indicated the presence of the test construct, which was confirmed by PCR using primers from apl-1 and the vector backbone of the rescue construct. A line was considered rescued only if the homozygous apl-1 animal carrying the putative rescue construct produced >25 viable progeny in each generation. Injection of the rol-6 or sur-5::GFP marker alone did not rescue the apl-1 lethality.

Construction of Transcriptional Fusion Plasmids.

Because we were unable to localize APL-1 with anti-APL-1EXT and anti-APL-1CYTO antibodies, we used reporter constructs to examine apl-1 expression (see SI Text for details). Transgenic animals were examined by using a confocal microscope (Photo 2–1 BX50; Olympus, Melville, NY), and images were processed by using Photoshop and Illustrator (Adobe Systems, San Jose, CA). GFP-expressing cells were identified based on their position, morphology, and/or projection pattern; their most likely identifications are presented.

Behavioral and Morphological Analysis of Animals.

To analyze brood size, L4 animals were individually plated and allowed to develop into adults. The number of eggs laid and the number of progeny that hatched were counted.

For analysis of movement, L4 animals were individually plated and allowed to develop into adults overnight. Each animal was sampled three times and the mean value was recorded. To analyze movement in liquid, animals were placed into 100 μl of M9 physiological buffer and the number of thrashes per min was counted. To analyze movement on a solid surface, animals were placed on a lawn of bacteria, and the number of head bends per min was counted.

To analyze the timing of development, single L4 animals were plated and allowed to lay 10–20 eggs. The mothers were removed, and after 3 days at 20°C the number of progeny in each developmental stage was determined on the basis of their size and/or gonadal development.

The morphology of animals was examined with Nomarski optics under an Axioplan (Zeiss, Thornwood, NY) or confocal microscope (Photo 2–1 BX50; Olympus); pictures were only taken of animals that were alive, as monitored by pharyngeal pumping. Images were processed by using Adobe Systems Photoshop and Illustrator.

Abbreviation

APP

amyloid precursor protein.