APL-1, a Caenorhabditis Elegans Protein Related to the Human ␤-Amyloid Precursor Protein, Is Essential for Viability

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

Angela Hornstena, Jason Lieberthalb,c, Shruti Fadiab,d, Richard Malinse,f, Lawrence Hag, Xiaomeng Xug, Isabelle Daigleh, Mindy Markowitzb,i, Gregory O’Connora,j, Ronald Plasterkk, and Chris Lig,h,l

Programs in aMolecular Biology, Cell Biology, and Biochemistry and bBiochemistry and Molecular Biology, and Departments of eChemistry and hBiology, Boston University, 5 Cummington Street, Boston, MA 02215; gDepartment of Biology, City College of the City University of New York, 160 Convent Avenue, New York, NY 10031; and kHubrecht Laboratory, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands

Edited by Iva S. Greenwald, Columbia University, New York, NY, and approved December 8, 2006 (received for review May 15, 2006) Dominant mutations in the amyloid precursor protein (APP) gene are APP transgene (15). Expression of human APP in Drosophila associated with rare cases of familial Alzheimer’s disease; however, wing imaginal discs results in a blistered wing phenotype, the normal functions of APP and related proteins remain unclear. The showing that overexpression of APP can disrupt cell adhesion in nematode Caenorhabditis elegans has a single APP-related gene, the transgenic animals (16). apl-1, that is expressed in multiple tissues. Loss of apl-1 disrupts In this article, we examine the role of apl-1 in C. elegans. several developmental processes, including molting and morphogen- Zambrano et al. (17) have reported mild pharyngeal defects when esis, and results in larval lethality. The apl-1 lethality can be rescued apl-1 activity is decreased by dsRNA-mediated interference by by neuronal expression of the extracellular domain of APL-1. These feeding. We genetically inactivated apl-1 and found that, like the data highlight the importance of the extracellular domain of an APP mammalian APP gene family, apl-1 has an essential role in C. family member and suggest that APL-1 acts noncell-autonomously elegans. In particular, APL-1 is necessary for proper molting and during development. Overexpression of APL-1 also causes several morphogenesis. Furthermore, expression of the extracellular do- defects, including a high level of larval lethality. Decreased activity of main of APL-1 in neurons is sufficient to rescue the apl-1 lethality. sel-12,aC. elegans homologue of the human ␥-secretase component These data highlight the significance of the extracellular domain of presenilin 1, partially rescues the lethality associated with APL-1 APL-1 and perhaps other APP-related proteins. overexpression, suggesting that SEL-12 activity regulates APL-1 activity either directly or indirectly. Results and Discussion apl-1 Is Expressed in Multiple Cell Types. apl-1 (C42D8.8) maps to Alzheimer’s disease ͉ genetics ͉ model system 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 lzheimer’s Disease (AD) is a progressive neurodegenerative determine the expression pattern of apl-1, we generated animals Adisorder that is characterized pathologically by the accu- carrying transcriptional and translational GFP reporter trans- mulation of dense plaques in the brains of AD patients. The main ␤ genes. All transgenic lines exhibited comparable expression component of these plaques is the -amyloid peptide (1, 2), patterns. Similar to the widespread expression of mammalian which is a cleavage product of the amyloid precursor protein APP (for review see ref. 18), apl-1 expression was detected in (APP; ref. 3). Autosomal dominant mutations in APP have been Ͼ50 neuronal, muscle, hypodermal, and supporting cells in correlated with a small number of early-onset AD cases (see adults [Fig. 2 and supporting information (SI) Fig. 4]. The larval Alzheimer’s Disease Mutation Database at www.molgen.ua. expression pattern was similar to the adult pattern with a few ac.be/ADMutations). Although APP has been implicated in exceptions; for instance, apl-1 is expressed in more ventral cord NEUROSCIENCE several processes in vitro, such as neurite outgrowth, cell adhe- motor neurons in the first larval stage (L1) animals than in other sion, and cell survival (for review see ref. 4), the in vivo functions larval stages or adults (data not shown). of APP remain unclear. Determining the in vivo functions of APP in mammals is com- apl-1 Is Essential for Viability. To determine the function of apl-1, plicated by the presence of two APP-related genes, APLP1 and we isolated a strain (pk53 apl-1:Tc1) containing a Tc1 transposon 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 Author contributions: A.H. and C.L. designed research; A.H., J.L., S.F., R.M., L.H., X.X., G.O., contain the ␤-amyloid peptide (5). Mice in which APP, APLP1,or and C.L. performed research; I.D., M.M. and R.P. contributed new reagents/analytic tools; APLP2 is inactivated are viable and have minor behavioral and A.H., J.L., S.F., R.M., L.H., X.X., I.D., and C.L. analyzed data; and C.L. wrote the paper. growth deficits (6–8). However, inactivation of APLP2 and either The authors declare no conﬂict of interest. APP or APLP1 results in early postnatal lethality (6, 8), indicating This article is a PNAS direct submission. that the APP family is essential for viability. The brains of double Abbreviation: APP, amyloid precursor protein. knockout animals exhibit no obvious morphological defects (6, 8). cPresent address: Department of Microbiology, New York University School of Medicine, By contrast, animals in which the entire APP gene family is New York, NY 10016. inactivated show cortical dysplasia and type 2 lissencephaly, indi- dPresent address: Robert Wood Johnson Medical School, Piscataway, NJ 08854. cating that the APP gene family is necessary for neurodevelopment fPresent address: Department of Pharmacology, Oxford University, Oxford OX1 3QT, and adhesion (9). United Kingdom. Although no APP gene has been identified in Drosophila iPresent address: Department of Medicine, Lenox Hill Hospital, New York, NY 10021. melanogaster or Caenorhabditis elegans, each organism contains jPresent address: Novartis Institutes for Biomedical Research, Cambridge, MA 02139. a single APP-related gene (10, 11). Inactivation of the Drosophila lTo whom correspondence should be addressed. E-mail: [email protected]. APP-related gene, Appl, causes abnormal synaptic differentia- This article contains supporting information online at www.pnas.org/cgi/content/full/ tion (12), axonal transport (13, 14), and phototactic behavior 0603997104/DC1. (15), the latter of which can be partially rescued with a human © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603997104 PNAS ͉ February 6, 2007 ͉ vol. 104 ͉ no. 6 ͉ 1971–1976 Downloaded by guest on September 27, 2021 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)InynIs79 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␮m; B,10␮m.)

Fig. 1. Molecular characterization of apl-1 alleles. (A) Schematic of the apl-1 genomic locus. The gray boxes represent the regions encoding the extracel- insertion in the apl-1 gene (Fig. 1A) and screened for imprecise lular domain of APL-1, the striped box indicates the transmembrane (TMD) transposon excisions that deleted parts of the apl-1 coding Ј and cytoplasmic domains, and the open box indicates the 3 UTR. apl-1 is region. We isolated two deletion mutants: yn5 (see below) and 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 dele- yn10 (Fig. 1A and SI Text). Because yn10 mutants produced no tions are indicated (see SI Text for exact lesions). AMB, OCH, and OPA indicate detectable APL-1 protein (Fig. 1C), we concluded that yn10 is a amber, ochre, and opal stop codons, respectively. (B) Schematic of proteins null allele. To isolate additional apl-1 alleles, we performed F1 encoded by constructs tested for rescue of the apl-1 lethality. The signal noncomplementation screens and isolated five more alleles: sequence (circles), extracellular E1 (amino acids 1–209) and E2 (amino acids yn23, yn29, yn30, yn31, and yn32. All of the isolated alleles ␣ 236–423) domains, TMD (gray box), cytoplasmic domain (C), and putative Go correspond to point mutations within the coding region of the 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. extracellular domain and cause phenotypes similar to yn10 (Fig. The number of lines that rescued the given apl-1 mutation relative to the total 1A and SI Text). number of independent transgenic lines is shown. ND, not determined. (C) apl-1(yn10) and the isolated point mutations cause a recessive yn10, yn23, and yn32 are likely null alleles. To maintain apl-1 homozygotes, all larval lethal phenotype. To determine when apl-1 is required, we lines carried a rescue construct (listed in brackets below but not shown) except observed the development of yn10 animals. Most mutants were yn5. All Western blots were probed with an antibody against the entire APL-1 morphologically WT at hatching. Shortly after hatching, how- 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 Extracts from WT animals contain full-length and glycosylated APL-1 (arrow- the rescue construct. No full-length APL-1 (arrowhead in top blot) or protein head; 105–110 kDa), a cleaved extracellular fragment sAPL-1 (open dot; Ϸ85 corresponding to in vitro-translated yn10 protein (yn10IV) (star in second blot) kDa), and high molecular mass forms that are presumably dimers (asterisk; was detected. Homozygous yn5 deletion mutants are viable and produce only Ϸ200–220 kDa). yn5 extracts contain only APL-1EXT (dot; Ϸ90 kDa), which is APL-1EXT (dot in top blot). yn23 dpy-8;[APL-1::GFP] animals (labeled yn23) slightly larger than sAPL-1 because it does not appear to be further cleaved by only contained APL-1::GFP (double arrowhead) and its cleavage product, secretases. Transgenic apl-1 overexpression lines (ynIs79 APL-1::GFP, ynIs86 sAPL-1 (open dot) from the rescue construct (third blot). No full-length APL-1 APL-1, ynIs13 nAPL-1, and ynIs12 nAPL-1) are in a WT or sel-12 ␥-secretase (arrowhead in third blot) or protein corresponding to in vitro-translated yn23 mutant background. ynIs79 APL-1::GFP animals contain proteins correspond- protein (star in fourth blot) was detected. yn5IV is shown as a size control. yn32 ing to sAPL-1 (open dot), APL-1::GFP (double arrowheads; Ϸ135 kDa), and is a missense mutation predicted to allow full-length APL-1 production. How- high molecular mass forms (double asterisk; Ϸ260 kDa) that are presumably ever, extracts from lon-2 yn32;[APL-1EXT] (labeled yn32) only contained dimers of APL-1::GFP; the high levels of presumptive dimers may be caused by APL-1EXT from the rescue construct (dot in bottom blot). No full-length APL-1 high levels of APL-1 or because the GFP tag promotes dimerization. The (arrowhead in bottom blot) or cleaved sAPL-1 (open dot in bottom blot) was endogenous WT protein is not detectable at this concentration of total detected. Extracts from yn5 animals were run as a size control. Note that protein in ynIs79 APL-1::GFP animals. Other transgenic animals contain full- APL-1EXT is slightly larger than sAPL-1. Molecular mass markers and estimated length and glycosylated APL-1 (arrowhead), sAPL-1 (open dot), and low levels weights are in kDa. (D) Expression of high levels of APL-1 in yn5 and integrated of presumptive dimers of APL-1 (asterisk). WT and yn5 in vitro-translated trangenic apl-1 strains in a WT or decreased sel-12 ␥-secretase background. proteins are shown as size controls; smaller IV proteins are presumably caused APL-1 expression driven by a pan-neuronal promoter is indicated by nAPL-1. by initiation from inappropriate downstream methionine codons. Molecular Note that the amount of total protein loaded on the Western blot (indicated mass markers are in kDa. Quantiﬁcation of APL-1 levels is in SI Table 2. See SI at the bottom) is varied up to 20-fold to allow visualization of WT protein. Text for details about methods.

1972 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603997104 Hornsten et al. Downloaded by guest on September 27, 2021 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 extrachro- mosomal 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 ϭ 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 Fig. 3. Either loss or overexpression of apl-1 causes morphological defects. isolated from all three mutants (data not shown). However, no (A and B) Head (A) and gonadal (B) regions of a WT ﬁrst larval stage (L1) endogenous mutant APL-1 protein was detected in any of the hermaphrodite animal. (C–F) Homozygous yn10 hermaphrodites in a sem-4;lon-2 background. The sem-4 mutation, which causes adult hermaph- three strains (Fig. 1C), indicating that these three mutations are rodites to retain their eggs, was used to aid in the observation of embryos. likely null alleles. The yn32 mutation causes a glutamic acid to Similar phenotypes were seen in a non-sem-4 background. (C) An L1 animal in lysine substitution at residue 372 in the E2 domain. In human which posterior morphogenesis was disrupted. The midregion of the animal APP the corresponding residue is not predicted to be important is shown. Posterior organs did not develop and the midregion ends abruptly for the structural stability of E2 (20); however, this residue is (arrows); the cuticle, however, extended posteriorly. Vacuoles (double arrows) proposed to be part of an ␣-helix that is necessary for APP can be seen throughout the animal. (D) A late L1/early L2 animal that showed cleavage (21). Animals carrying an apl-1(yn32)::GFP transgene large vacuoles (double arrows) is shown. (E) A late L1/early L2 animal in which express GFP faintly (data not shown), suggesting that yn32 a large gap between the gonad and overlying intestine was seen (arrow). (F) animals die from insufficient levels of APL-1::yn32 protein 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 because it is unstable, degraded quickly, or not cleaved to (double arrow). (G and H)AnL1apl-1 overexpression animal (ynIs79 produce sAPL-1. APL-1::GFP) had a translucent appearance. Cells and organs appeared in stark To address whether human APP is a functional homologue of relief, particularly in the midregion of the animal (H). Arrows point to muscle apl-1, we determined whether human APP expressed using apl-1 nuclei. Anterior is to the left. All micrographs were taken on live animals; regulatory sequences could rescue the apl-1 mutants. This construct except for WT, none of the animals were anesthetized. ph, ﬁrst bulb of was unable to rescue yn10 (0/20 rescued lines). We have not pharynx, nr, nerve ring region, g, gonad, int, intestine. (Scale bars: 10 ␮m.) determined whether other human APP, APLP1, APLP2, or Dro- sophila Appl constructs are able to rescue apl-1 mutants. ever, the mutants (35/41 animals) began to accumulate variously The apl-1-Induced Lethality Is Not Caused by Activation of Cell Death sized vacuoles (Fig. 3 C, D, and F) that appeared to be within the NEUROSCIENCE Pathways. Mammalian APP has been proposed to regulate large syncytial hypodermal cells. A few mutants (4/30 animals) apoptosis (22), yet APP can also be cleaved by different caspases arrested or died during L1 because of severe morphological (23–25). We, therefore, examined whether the apl-1 lethality is defects (Fig. 3C). A small number of L1 animals (3/30 animals) caused by activation of an apoptotic or necrotic cell death had large gaps between organs, perhaps because of an adhesion pathway. In C. elegans ced-3 encodes a caspase that is essential or osmoregulatory defect (Fig. 3E). As monitored by a neural- for execution of apoptosis (26) and crt-1 encodes calreticulin, specific GFP marker, the nervous system of L1 mutants ap- which is essential for execution of necrotic cell deaths (27). peared normal and axon bundles were correctly positioned (data Neither loss of ced-3 caspase nor loss of crt-1 calreticulin activity not shown). No other phenotype was seen until the first to rescued the yn10 lethality, indicating that the apl-1 lethality is not second larval stage (L2) transition when all mutant animals caused by ectopic activation of either cell death pathway. displayed a molting defect. During each larval stage transition, WT animals synthesize a new cuticle and shed their old cuticle. Expression of the Extracellular Domain of APL-1 Is Sufficient for The mutants synthesized a new L2 cuticle but failed to shed their Viability. Different domains of human APP have been shown to old L1 cuticle, thereby becoming trapped within the old cuticle interact with a number of proteins in vitro (for reviews see refs. and dying (Fig. 3F). Hence, inactivation of apl-1 disrupts molting 28 and 29). For example, the APP extracellular domain interacts and morphogenesis. with Notch (30), low-density lipoprotein receptor-related pro- To confirm that these phenotypes were caused by loss of tein (31), and components of the extracellular matrix (4, 28, 29). apl-1, we transformed apl-1 heterozygotes with an 8.45-kb apl-1 Similarly, defined regions of the APP cytoplasmic domain genomic fragment and recovered transgenic homozgyous apl-1 interact with several proteins, such as Go␣, Disabled, and progeny that were rescued for all apl-1 mutant phenotypes (SI kinesin-1 (14, 28, 29); in addition, the APP cyptoplasmic tail, Fig. 5). By contrast, the same genomic fragment containing point when complexed with Fe65, has been proposed to enter the mutations in the coding region failed to rescue the apl-1 lethality nucleus to affect transcription (32). The biological significance (SI Fig. 5). Expression of an APL-1::GFP fusion protein using of these interactions, however, remains unclear. apl-1 regulatory sequences also rescued the lethality (Fig. 1B). To determine which domains of APL-1 are necessary for Together, these data indicate that the observed phenotypes function in vivo, we tested whether apl-1 transgenes that were

Hornsten et al. PNAS ͉ February 6, 2007 ͉ vol. 104 ͉ no. 6 ͉ 1973 Downloaded by guest on September 27, 2021 Table 1. Overexpression of apl-1 causes decreased brood size and sluggishness Brood size Movement

No. of thrashes No. of head bends Genotype No. of eggs No. of adult progeny % survival per min per min

WT 250.6 Ϯ 5.3 (n ϭ 46) 261.6 Ϯ 5.0 (n ϭ 46) 100 86.4 Ϯ 2.7 (n ϭ 60) 15.5 Ϯ 0.7 (n ϭ 60) yn5 APL-1EXT 187.9 Ϯ 4.9* (n ϭ 33) 192.2 Ϯ 5.0* (n ϭ 33) 100 77.5 Ϯ 5.0 (n ϭ 30) 14.0 Ϯ 0.9 (n ϭ 40) Transgenic apl-1 overexpression lines† ynIs86 APL-1 203.8 Ϯ 8.7* (n ϭ 43) 192.6 Ϯ 7.0* (n ϭ 43) 94.5 56.0 Ϯ 3.0* (n ϭ 30) 11.8 Ϯ 0.8 (n ϭ 44) ynIs79 217.6 Ϯ 7.9 (n ϭ 31) 64.6 Ϯ 5.1* (n ϭ 31) 29.7 32.9 Ϯ 3.3* (n ϭ 31) 6.9 Ϯ 0.6* (n ϭ 39) APL-1::GFP ynIs13 nAPL-1 228.3 Ϯ 4.8 (n ϭ 43) 229.7 Ϯ 5.0* (n ϭ 43) 100 52.0 Ϯ 3.2* (n ϭ 30) 11.8 Ϯ 0.9 (n ϭ 40) ynIs13; ynIs86 209.8 Ϯ 4.4* (n ϭ 50) 197.3 Ϯ 4.2 (n ϭ 50) 94.0 16.1 Ϯ 1.4* (n ϭ 31) 5.4 Ϯ 0.6* (n ϭ 40) Decreased sel-12 ␥-secretase activity in apl-1 overexpression lines sel-12 103.2 Ϯ 6.2* (n ϭ 48) 112.8 Ϯ 7.3* (n ϭ 48) 100 84.2 Ϯ 1.9 (n ϭ 20) 18.9 Ϯ 1.9 (n ϭ 19) ␥-secretase sel-12 yn5 86.3 Ϯ 10.4* (n ϭ 43) 90.9 Ϯ 0.9.9* (n ϭ 43) 100 73.2 Ϯ 3.3*‡ (n ϭ 40) 12.6 Ϯ 0.9* (n ϭ 40) sel-12;ynIs79 103.4 Ϯ 8.5* (n ϭ 56) 72.8 Ϯ 6.1* (n ϭ 56) 70.4 11.8 Ϯ 1.1*‡ (n ϭ 20) 8.4 Ϯ 1.0*‡ (n ϭ 19) sel-12 ynIs86 54.7 Ϯ 7.0*‡ (n ϭ 19) 59.1 Ϯ 7.8*‡ (n ϭ 19) 100 35.7 Ϯ 5.3*‡ (n ϭ 20) 11.4 Ϯ 0.9‡ (n ϭ 20)

Values represent the means Ϯ 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 Ͻ 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 Ͻ 0.01; one-way ANOVA; Newman–Keuls post hoc test).

deleted for specific domains were sufficient for rescue of the (nAPL-1; refs. 33 and 34) was sufficient to rescue the apl-1 apl-1 lethality. As much research has focused on interactions of lethality. Furthermore, expression of APL-1EXT in neurons the cytoplasmic domain of mammalian APP, we first investigated with the snb-1 promoter also rescued the apl-1 lethality (6/6 yn10 whether cytoplasmic domains within APL-1 were needed for and 1/1 yn32 rescued lines), suggesting that cleavage of full- viability. Deletion of the putative Go␣ binding site (11) did not length APL-1 to release sAPL-1 from neurons is sufficient for affect transgene rescue of the apl-1 lethality (Fig. 1B). Strikingly, proper molting and morphogenesis. The failure to rescue by an APL-1 protein lacking the transmembrane and cytoplasmic hypodermal expression of APL-1 was puzzling, because sAPL-1 domains also gave complete rescue (Fig. 1B), indicating that the is presumably affecting the hypodermal cells to influence molt- activities necessary for rescue are contained within the extra- ing. However, hypodermal cells might lack the appropriate cellular domain of APL-1. Consistent with this result, apl-1(yn5) proteases to cleave APL-1 to generate sAPL-1. deletion mutants are homozygous viable and produce only the extracellular domain of APL-1 (APL-1EXT) (Fig. 1D), which is Overexpression of APL-1 Causes Defects in Brood Size, Movement, and slightly larger than the WT cleaved APL-1 (sAPL-1). These data Viability. To determine whether APL-1 overexpression caused argue that the extracellular domain is critical for APL-1 rescuing defects, we analyzed several transgenic lines in which the apl-1 activity and that the lethality is not caused by the lack of either rescuing transgenes were integrated into the genome and crossed protein interactions with the cytoplasmic domain of APL-1 or into a WT background. These strains include ynIs86, which apl-1-dependent transcription. contains integrated copies of APL-1; ynIs79, which contains To determine which regions of the APL-1 extracellular do- integrated copies of APL-1::GFP; and ynIs12 and ynIs13, which main are necessary for rescue, we deleted sequences encoding contain integrated copies of nAPL-1. By Western blot analysis, the E1 domain (⌬E1), the E2 domain (⌬E2), or the E1 through APL-1 or APL-1::GFP (which will be referred collectively as most of the E2 domains (⌬E1-E2) and tested the modified APL-1 henceforth) levels were at least 15-fold higher in the constructs for rescuing activity. Both APL-1⌬E1 and APL-1⌬E2 transgenic overexpression lines compared with WT (Fig. 1D and were each able to rescue apl-1 mutants, whereas APL-1⌬E1-E2 SI Table 2). A similar increase in the level of APL-1EXT was also did not (Fig. 1B). Thus, despite structural dissimilarities, either observed in apl-1(yn5) mutants (Fig. 1D and SI Table 2). This the E1 or E2 domain by itself is sufficient for viability and the increased APL-1EXT level in yn5 mutants may be caused by two domains function independently and redundantly. The E1 constitutive release of APL-1EXT or increased stability and/or and E2 domains of mammalian APP have been shown to bind resistance to the degradation of APL-1EXT. heparin, collagen, and laminin, presumably to mediate cell–cell Transgenic strains overexpressing APL-1 had defects in brood or cell–substratum adhesion or increase neurite outgrowth (28, size, movement, and viability; the severity of these defects was 29); APL-1 may similarly bind the extracellular matrix to mediate strongly correlated with the level of APL-1 overexpression equivalent functions. (Table 1, SI Table 2, and Fig. 1D). Wild-type animals generally lay between 250 to 300 eggs (ref. 35 and Table 1). Animals apl-1 Expression in Neurons Is Sufficient for Viability. To determine overexpressing APL-1 laid significantly fewer eggs than WT the site of action of apl-1, we expressed full-length APL-1::GFP (Table 1). Because apl-1(yn5) APL-1EXT animals also showed by using different tissue-specific promoters. Expression of a decreased number of progeny (Table 1), we hypothesize that APL-1 in pharyngeal muscle, body wall muscles, or hypodermal brood size is decreased by elevated levels of cleaved APL-1. The cells failed to rescue the apl-1(yn10) lethality (0/4, 0/6, and 0/3 increased level of extracellular APL-1 may interfere with cell– rescued lines, respectively). By contrast, APL-1 expression cell interactions, thereby disrupting morphogenesis and/or go- driven by either the pan-neuronal snb-1 (3/3 yn10 and 1/1 yn23 nadal development. rescued lines) or rab-3 (4/4 rescued yn10 lines) promoters Transgenic animals overexpressing APL-1 also had subtle

1974 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603997104 Hornsten et al. Downloaded by guest on September 27, 2021 movement defects, which we quantified in two motor assays. does not appear to affect the ␣-secretase cleavage of APL-1. We When placed into physiological buffer, WT animals begin to propose that SEL-12, like mammalian PS1, either directly or swim, and the number of body flexures or thrashes per minute indirectly regulates levels and/or trafficking of APL-1 and a can be quantified (Table 1). All apl-1 transgenic overexpression change in this regulation affects the viability of the animals. strains showed a significantly reduced thrashing rate (Table 1). Second, on a solid surface WT animals move in a sinusoidal Concluding Remarks waveform, which can be quantified by counting the number of Our results indicate that APL-1 is an essential, multifunctional head bends per min. All transgenic overexpression strains had a protein involved in molting, reproduction, locomotion, and decreased head bend rate compared with WT (Table 1). Fur- morphogenesis. Our finding that release of APL-1EXT from thermore, a transgenic line that was homozygous for two inte- neurons is sufficient for viability highlights the importance of the grated transgenes produced higher levels of APL-1 (Fig. 1D and extracellular domain of an APP-related protein and suggests that SI Table 2) and was more sluggish than either parental line alone the source of the cleaved APL-1 fragment sAPL-1 and its spatial (Table 1). Increasing levels of APL-1, therefore, inhibit move- distribution are critical. We suggest that the extracellular do- ment, perhaps by interfering with motor neuron functions. mains of mammalian APP and related proteins may also have Several APL-1 overexpressing strains, particularly apl-1(yn5) critical functions in the nervous system. APL-1EXT animals, developed more slowly than WT (SI Table 3). Wild-type animals lay eggs that hatch and develop into adults Materials and Methods in Ϸ3 days at 20°C (SI Table 3). By contrast, after 3 days, the Strains. C. elegans strains were grown at 20°C and maintained majority of apl-1(yn5) APL-1EXT progeny were still in a late according to Brenner (40). The mutations used are as described larval stage (SI Table 3). Transgenic overexpression animals also in Wormbase (www.wormbase.org) and include: LGI, sem- showed varying degrees of developmental delay (SI Table 3), 4(n1378); LGV, crt-1(ok948); and LGX, lon-2(e678), dpy- suggesting that higher levels of APL-1 and/or APL-1EXT inter- 8(e130), lin-15(n765), sel-12(ar131). Integrated transgenic lines fere with the normal developmental progression of the animal. were: LGIII, ynIs12 (nAPL-1; Psnb-1::apl-1); LGV, ynIs79 The ynIs79 APL-1::GFP overexpression line exhibited the most APL-1::GFP, ynIs13 (nAPL-1; Psnb-1::apl-1); and LGX, severe phenotypes and expressed the highest levels of APL-1 ynIs86 APL-1. Nonintegrated transgenic lines used were: (Ϸ180-fold higher than WT; Fig. 1D and SI Table 2). Interestingly, ynEx106 APL-1EXT, ynEx106A APL-1EXT, ynEx165 although ynIs79 APL-1::GFP overexpression animals laid a similar (Psnb-1::APL-1EXT), and ynEx166 (Psnb-1::APL-1EXT). CL1093 number of eggs compared with other transgenic overexpression (Papl-1::GFP) was a kind gift of Chris Link (University of lines, Ϸ70% of the animals died during L1 (Table 1), and this Colorado, Boulder, CO). lethality rate was unchanged in a ced-3 caspase loss-of-function background (data not shown). No such lethality was associated with Identification of apl-1 Alleles. All isolated mutants were back- apl-1(yn5) or other transgenic overexpression lines, except ynIs86 crossed, and molecular lesions were determined by DNA se- APL-1 animals, which showed a low lethality rate (Table 1). ynIs79 quencing. pk53 (apl-1::Tc1) was isolated by screening a mutant APL-1::GFP overexpression animals appear morphologically WT bank of transposon-insertion strains (41). The yn5 and yn10 at hatching. At variable times during L1, ynIs79 APL-1::GFP mutations were isolated in a PCR-based screen of 2,340 pk53 overexpression animals become translucent and large gaps are (apl-1::Tc1) populations (41). F1 noncomplementation screens present between organs (Fig. 3 G and H). Whereas in WT animals were used to isolate yn23, yn29, yn30, yn31, and yn32 from a total muscle bundles appear contiguous with intestinal and gonadal cells, of 3,105 mutagenized haploid genomes (see SI Text). in ynIs79 APL-1::GFP overexpression animals muscle bundles are clearly separated from intestinal cells and the gonad when visual- Construction and Microinjection of Test Plasmids. Several derivatives ized by Nomarski optics (Fig. 3H). When visualized by fluorescently of the cosmid C42D8, including two with point mutations in the tagged phalloidin, the fibers within the muscle bundles of ynIs79 apl-1 coding region, were tested for rescue (SI Fig. 5 and SI Text). APL-1::GFP overexpression animals were difficult to visualize, as Constructs containing different deletions in apl-1 were gener- though the muscle bundles were condensed (data not shown). ated by PCR and sequenced to verify the deletions (see SI Text). NEUROSCIENCE These phenotypes are consistent with disruptions in cell adhesion, Heterologous promoters used to drive apl-1 cDNA expression whereby elevated expression of APL-1 interferes with the normal were: the pan-neuronal promoters synaptobrevin (snb-1) (33) adhesion contacts between cells, or in osmoregulation. and rab-3 (34); the pharyngeal and body wall muscle promoters myo-2 and myo-3, respectively (42); and the hypodermal cell Loss of sel-12 Presenilin Partially Rescues the Lethality Caused by apl-1 col-10 promoter (43). Constructs were microinjected at 50–150 Overexpression. Presenilin 1 (PS1) has been proposed to be part ng/␮l; dominant markers (50 ng/␮l) coinjected to identify trans- of the ␥-secretase complex that cleaves substrates such as human genic animals were pRF4 rol-6 (44), pTG96 sur-5::GFP (45), or APP and the LIN-12/Notch receptor (for review see ref. 36); in pJM24 lin-15 (46). GFP-tagged constructs were generally not addition, PS1 may regulate the level, amount of cleavage, and/or coinjected with marker plasmids. The transgenic arrays were not trafficking of APP (37, 38). One C. elegans homologue of PS1 is integrated into the genome unless otherwise noted. sel-12, which was identified as a suppressor of a lin-12 gain-of- function mutation (39). If SEL-12 similarly regulates levels, Determination of apl-1 Rescue. Test constructs were coinjected into processing, and/or trafficking of APL-1, the amount of lethality heterozygous lon-2 apl-1(yn10)/dpy-8, apl-1(yn23) dpy-8/lon-2,or seen in ynIs79 APL-1::GFP overexpression animals might be lon-2 apl-1(yn32)/dpy-8 animals with the marker plasmids pRF4 altered by loss of sel-12 activity. Reduced sel-12 activity causes rol-6 (44) or pTG96 sur-5:GFP (45) unless the construct was an egg-laying defect that decreases the number of eggs (39), but GFP-tagged. The progeny of the injected animals were tested for does not affect the viability of the progeny (Table 1). The rescue as follows. The presence of adult progeny with the lethality of ynIs79 APL-1::GFP overexpression was partially phenotype caused by the genetic marker in cis to apl-1 (i.e., Lon rescued (30% compared with 70% lethality) by reduced sel-12 or Dpy) suggested rescue by the injected transgene. These activity (Table 1). However, all forms of APL-1, including transgenic progeny were individually plated and allowed to cleaved, full-length, and high-molecular-weight forms of APL-1, self-fertilize. Confirmation that the strain was homozygous for were present in ynIs79 APL-1::GFP; sel-12 animals in roughly the the apl-1 allele was done by PCR, PCR followed by restriction same proportions but at slightly lower levels than in ynIs79 digests, and/or DNA sequence analysis. The presence of GFP or APL-1::GFP animals alone (Fig. 1D and SI Table 2). Thus, sel-12 the coinjection marker (i.e., rol-6 or sur-5::GFP) indicated the

Hornsten et al. PNAS ͉ February 6, 2007 ͉ vol. 104 ͉ no. 6 ͉ 1975 Downloaded by guest on September 27, 2021 presence of the test construct, which was confirmed by PCR The morphology of animals was examined with Nomarski optics using primers from apl-1 and the vector backbone of the rescue under an Axioplan (Zeiss, Thornwood, NY) or confocal micro- construct. A line was considered rescued only if the homozygous scope (Photo 2–1 BX50; Olympus); pictures were only taken of apl-1 animal carrying the putative rescue construct produced animals that were alive, as monitored by pharyngeal pumping. Ͼ25 viable progeny in each generation. Injection of the rol-6 or Images were processed by using Adobe Systems Photoshop and sur-5::GFP marker alone did not rescue the apl-1 lethality. Illustrator.

Construction of Transcriptional Fusion Plasmids. Because we were We thank Karen Thijssen for help with isolating pk53; Laurie Nelson for unable to localize APL-1 with anti-APL-1EXT and anti-APL- RT-PCR results; Kyuhyung Kim for help with cell identifications; Mark 1CYTO antibodies, we used reporter constructs to examine apl-1 Schomer for excellent technical support; Victor Ambros (Dartmouth Col- expression (see SI Text for details). Transgenic animals were lege, Hanover, NH), Alan Coulson (Sanger Centre, Cambridge, UK), examined by using a confocal microscope (Photo 2–1 BX50; Monica Driscoll (Rutgers University, Piscataway, NJ), Andy Fire (Stanford Olympus, Melville, NY), and images were processed by using University, Stanford, CA), Iva Greenwald, Min Han (University of Colo- Photoshop and Illustrator (Adobe Systems, San Jose, CA). rado, Boulder, CO), Bob Horvitz (Massachusetts Institute of Technology, GFP-expressing cells were identified based on their position, Cambridge, MA), Jim Kramer (Northwestern University Medical School, morphology, and/or projection pattern; their most likely identi- Chicago, IL), Brian Onken (Rutgers University), Chris Link, Lavanya fications are presented. Muthukumar (Boston University), Mike Nonet (Washington University, St. Louis, MO), Evgeny Rogaev (University of Massachusetts, Worcester, Behavioral and Morphological Analysis of Animals. To analyze brood MA), Dennis Selkoe (Harvard Medical School, Boston, MA), and Paul size, L4 animals were individually plated and allowed to develop Sternberg (California Institute of Technology, Pasadena, CA) for strains, into adults. The number of eggs laid and the number of progeny that vectors, and plasmids; Scott Emmons (Albert Einstein College of Medicine, hatched were counted. Bronx, NY) for a C. elegans genomic library; Alan Coulson for positioning For analysis of movement, L4 animals were individually plated the apl-1 genomic clone on the physical map; and Phil Anderson, John and allowed to develop into adults overnight. Each animal was Celenza, Scott Clark, John Collins, Jean-Charles Epinat, Chip Ferguson, sampled three times and the mean value was recorded. To analyze Piali Sengupta, and members of C.L.’s laboratory (past and present) for helpful discussions and/or comments on the manuscript. Some strains in this movement in liquid, animals were placed into 100 ␮lofM9 work were provided by the Caenorhabditis Genetics Center, which is funded physiological buffer and the number of thrashes per min was by the National Institutes of Health National Center for Research Re- counted. To analyze movement on a solid surface, animals were sources. This work was supported by National Institutes of Health Grants placed on a lawn of bacteria, and the number of head bends per min AG00708 and AG11875 (to C.L.), the American Health Assistance Foun- was counted. dation (C.L.), the Alzheimer’s Association (Willard and Rachel Olsen Pilot To analyze the timing of development, single L4 animals were and Investigator Research Grants) (C.L.), National Institutes of Health plated and allowed to lay 10–20 eggs. The mothers were removed, Research Centers in Minority Institutions Grant RR03060 (to the City and after 3 days at 20°C the number of progeny in each develop- College of the City University of New York), the Arnold and Mabel mental stage was determined on the basis of their size and/or Beckman Foundation (R.M. and M.M.), and the Boston University Un- gonadal development. dergraduate Research Opportunities Program (S.F. and J.L.).

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1976 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603997104 Hornsten et al. Downloaded by guest on September 27, 2021