Alzheimer‐related protein APL‐1 modulates lifespan through heterochronic gene regulation in Caenorhabditis elegans

Summary Alzheimer's disease (AD) is an age‐associated disease. Mutations in the amyloid precursor protein (APP) may be causative or protective of AD. The presence of two functionally redundant APP‐like genes (APLP1/2) has made it difficult to unravel the biological function of APP during aging. The nematode Caenorhabditis elegans contains a single APP family member, apl‐1. Here, we assessed the function of APL‐1 on C. elegans’ lifespan and found tissue‐specific effects on lifespan by overexpression of APL‐1. Overexpression of APL‐1 in neurons causes lifespan reduction, whereas overexpression of APL‐1 in the hypodermis causes lifespan extension by repressing the function of the heterochronic transcription factor LIN‐14 to preserve youthfulness. APL‐1 lifespan extension also requires signaling through the FOXO transcription factor DAF‐16, heat‐shock factor HSF‐1, and vitamin D‐like nuclear hormone receptor DAF‐12. We propose that reinforcing APL‐1 expression in the hypodermis preserves the regulation of heterochronic lin‐14 gene network to improve maintenance of somatic tissues via DAF‐16/FOXO and HSF‐1 to promote healthy aging. Our work reveals a mechanistic link of how a conserved APP‐related protein modulates aging.


Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder affecting over five million Americans and is the sixth leading cause of death (Alzheimer's Association 2015). The etiology of Alzheimer's disease is unclear. The highest risk factor for developing Alzheimer's disease is age, whereby 96% of patients with AD develop the disease at 65 years or older (Alzheimer's Association 2015). The disease is characterized by the deposition of amyloid plaques in brain tissue; the major component of the plaques is the beta-amyloid peptide, a cleavage product of the amyloid precursor protein (APP) (reviewed Selkoe & Hardy, 2016). Mutations in APP and in genes that produce the presenilin enzymes that are part of the c-secretase complex that cleaves APP are correlated with cases of familial Alzheimer's disease (Tanzi, 2012;reviewed Selkoe & Hardy, 2016).
In mammals, the functional analysis of APP is complicated by the presence of two APP-related proteins, APLP1 and APLP2 (reviewed Shariati & De Strooper, 2013). Whereas knockouts of individual APP family members are viable, double knockouts of APP and APLP2 or APLP1 and APLP2 result in postnatal lethality (von Koch et al., 1997;Herms et al., 2000Herms et al., , 2004, indicating an essential role for the APP family and functional overlap among family members that is not dependent on the beta-amyloid peptide. Knockout of the entire APP gene family results not only in postnatal lethality, but also defects in neuronal migration and type II lissencephaly (Weyer et al., 2011). Interestingly, duplication of the APP gene has also been associated with families that develop early-onset Alzheimer's disease (Cabrejo et al., 2006;Rovelet-Lecrux et al., 2006;Sleegers et al., 2006). More strikingly, patients with Down syndrome, who have a trisomy of chromosome 21, have a high incidence of earlyonset Alzheimer's disease (Korbel et al., 2009;reviewed in Shariati & De Strooper, 2013). These findings suggest that higher levels of APP, whether it be higher levels of the beta-amyloid peptide, the extracellular or cytoplasmic fragments, and/or full-length APP, are correlated with early-onset Alzheimer's disease. However, the cellular functions of APP and related proteins remain unclear.
The functions of genes correlated with Alzheimer's disease, including the presenilin genes, were first identified in genetic screens using the nematode Caenorhabditis elegans [reviewed (Ewald & Li, 2010)]. We have been examining the role of APL-1, the C. elegans orthologue of mammalian APP. Similar to human APP, C. elegans APL-1 is a single transmembrane-spanning protein with a large extracellular and a small intracellular domain, both of which share sequence homology to human APP (Daigle & Li, 1993). Analogous to the postnatal lethality of APP family-knockout mice, loss of apl-1 in C. elegans results in a completely penetrant larval lethality that is rescued by the reintroduction of either full-length APL-1 or only the extracellular domain of APL-1 (Hornsten et al., 2007). This finding was later extended to mammals, whereby knockin of sAPPa rescued the postnatal lethality of the APP-APLP2double-knockout mice (Weyer et al., 2011). Furthermore, overexpression of APL-1 in C. elegans also causes an early developmental lethality, but the lethality shows an incomplete penetrance that is correlated with higher levels of APL-1 (Hornsten et al., 2007). Similarly, overexpression of human or mouse APP in mice causes early developmental lethality in the absence of amyloid plaques (Hsiao et al., 1995). This correlation suggests that findings in C. elegans can provide insights into the rudimentary conserved functions of APP (Ewald & Li, 2012).
Here, we examined the effects of overexpressing APL-1 during adulthood on lifespan in C. elegans. Overexpressing APL-1 in neurons shortens lifespan, whereas overexpressing APL-1 in hypodermis extends lifespan. This lifespan extension is independent from longevity mediated by reduced insulin/IGF-1 signaling (rIIS) or by reduced germ stem cell numbers (rGSC). However, genes that are essential for rIIS and rGSC longevity, such as the FOXO transcription factor DAF-16, heat-shock factor HSF-1, and vitamin D-like nuclear hormone receptor DAF-12, are required for APL-1-dependent longevity, suggesting that APL-1 interacts with these genes in parallel to rIIS and rGSC. We found that hypodermal APL-1 represses the transcriptional function of the heterochronic transcription factor LIN-14, which has been shown to regulate longevity through HSF-1 and DAF-16. Repressing LIN-14 during aging is required for hypodermal-expressed APL-1 to extend lifespan and higher levels of LIN-14 are sufficient to abrogate the APL-1-induced longevity. These findings suggest that APL-1 promotes longevity by regulating heterochronic processes.

Results
Ectopic expression of APL-1 during adulthood increases lifespan High levels of APL-1 cause larval lethality (Hornsten et al., 2007). To bypass the developmental effects of APL-1 and to determine the role of APL-1 during aging, we used transgenic animals (ynIs14) that have the hsp-16.2 promoter fused with an apl-1 cDNA [Phsp-16.2::APL-1; (Ewald et al., 2012a)] and induced APL-1 expression in adults in all tissues via heat shock. Surprisingly, a single heat shock for 3.5 hours in day one adults mildly increased lifespan (5%; Fig. 1a). Moreover, two hours of heat shock administered every second day (Fig. 1b) or applying moderate heat continuously ( Fig. 1c; 25°C) robustly increased lifespan (12-30%) of ynIs14 [Phsp-16.2::APL-1] animals compared to wild-type. As a control, the same animals were assayed in parallel at 15°C, a temperature at which the hsp-16.2 promoter is not active, and no change in lifespan was observed ( Fig. 1d; Table S1). These results suggest that inducing ubiquitous overexpression of APL-1 exclusively during adulthood is sufficient to extend lifespan.

Overexpressing APL-1 in neurons shortens lifespan
The finding that ubiquitous adulthood expression of APL-1 increased lifespan was surprising. To gain some mechanistic insights into how this lifespan extension is mediated, we investigated in which tissue apl-1 expression is necessary for lifespan extension. C. elegans expresses apl-1 strongly in neurons throughout its life cycle (Hornsten et al., 2007) and neurons regulate aging and longevity in a conserved spectrum of animals, including C. elegans (Kenyon, 2010). To investigate whether APL-1 expressed in neurons increases lifespan, we examined transgenic animals carrying multicopy chromosomal arrays where apl-1 is driven by different promoters: its endogenous promoter, the pan-neuronal rab-3 promoter that drives expression only in neurons, or the ceh-36 or mec-4 promoters that drive expression in a subset of neurons. Overexpression of APL-1 with its own promoter, which includes strong expression in neurons and glial cells ([Papl-1::apl-1::GFP]; Fig. 1e; Table S2), shortened lifespan. Similarly, overexpressing APL-1 in all neurons with the rab-3 (Prab-3::APL-1::GFP; Fig. 1f; Table S2), or a subset of sensory neurons (Pceh-36::APL-1::GFP; Fig. 1g and Pmec-4::APL-1::GFP; Fig. 1h; Table S1), also shortened lifespan. Collectively, these results indicate that neuronal overexpression of APL-1 shortens lifespan and is in stark contrast to the extended lifespan induced by heat-shock overexpression of APL-1 in all tissues, including neurons, in adults ( Fig. 1). Therefore, we hypothesized that expression of APL-1 in tissues other than neurons must activate a protective pathway to circumvent the detrimental effects of APL-1 expression in neurons.

Overexpression of functional APL-1 is required to increase lifespan
There are several possible mechanisms by which Psnb-1::apl-1 cDNA overexpression could increase lifespan. For example, lowering food intake results in caloric restriction that increases lifespan (Kenyon, 2010). However, pharyngeal pumping rates, a measure of food intake, were unaltered in young transgenic APL-1 animals (Fig. S1c). Another hallmark of caloric-restricted animals is lower fat content (Heestand et al., 2013). The fat content of long-lived Psnb-1::APL-1 animals was not lower compared to wild-type. Furthermore, the fat content of short-lived apl-1 (yn5) and Papl-1::APL-1 animals was also lower compared to wild-type animals ( Fig. S2), suggesting that the effects of APL-1 on fat content do not correlate with its effect on lifespan and are, therefore, uncoupled from longevity. Lifespan extension can also be induced in C. elegans by mobilizing the unfolded protein response (UPR) (Taylor & Dillin, 2013). APL-1 is a glycosylated, transmembrane protein that is presumably processed in the endoplasmic reticulum (ER). Overexpression of APL-1 could overload the ER causing ER stress, thereby activating the UPR. However, APL-1 overexpression does not cause ER stress or induce the unfolded protein response (Fig. S3a-c). The increased lifespan, however, was dependent on a functional APL-1 protein. The missense mutation apl-1(yn32) causes an APL-1 E371K substitution that presumably interferes with alpha secretase cleavage at the cell membrane. Expression of the apl-1(yn32) transgene did not rescue the apl-1(null) larval lethality (Hornsten et al., 2007;Hoopes et al., 2010), suggesting that APL-1(yn32) is not sufficient for rescue. Animals carrying the Psnb-1:: APL-1(E371K)::GFP transgene (ynEx236, 237, or 238) showed no lifespan extension (Fig. 2h), suggesting that only functional APL-1 can increase lifespan and that nonfunctional APL-1 has no adverse effects. To further exclude that any protein structurally similar to APL-1 (e.g., human APP) driven by the snb-1 promoter could extend lifespan, we measured the lifespan of animals carrying the Psnb-1::humanAPP transgene, which had no effect on lifespan ( Fig. 2c; Table S3). Human APP is unable to rescue the apl-1(null) larval lethality (Hornsten et al., 2007;Wiese et al., 2010), suggesting that human APP is not functional in C. elegans. Taken together, these results suggest that overexpression of functional APL-1 increases lifespan. APL-1 represses LIN-14 to promote healthy aging, C. Y. Ewald et al.  Table S1. For

APL-1 protein levels do not correlate with changes in lifespan
Another possibility for APL-1 lifespan effects is that levels of the APL-1 protein determine lifespan. However, levels of APL-1 protein did not correlate with changes in lifespan, as ynIs79 [Papl-1::apl-1::GFP] animals expressed the highest levels of APL-1::GFP, but showed a shortened lifespan (Figs. 1e and S3d-f). Instead, we postulate that the increased lifespan is context dependent; that is, where APL-1 is overexpressed determines the APL-1 effect on longevity. Hence, as long as APL-1 is overexpressed in the relevant tissues, lifespan will be increased. We tested this model by mating short-lived ynIs86 [Papl-1::APL-1] transgenic animals that show a 177-fold increase in APL-1 protein (Hornsten et al., 2007) with long-lived ynIs12 [Psnb-1::APL-1] transgenic animals that show a 71-fold increase in APL-1 protein (Hornsten et al., 2007). The APL-1 represses LIN-14 to promote healthy aging, C. Y. Ewald et al. resultant double transgenic strain showed a 199-fold increase in APL-1 levels compared to wild-type and was long-lived (Figs. 2i and S3d-e; Table S3), demonstrating that the tissue in which apl-1 is expressed and not the overall levels of APL-1 protein determines lifespan.

Discussion
Mammalian APP is expressed in all tissue types (Tanzi et al., 1987), but an unresolved question remains as to whether APP has a similar function in all cell types or whether its functions are context dependent. As this question is difficult to address in mammalian systems, we turned to C. elegans where the use of cell-specific promoters is routine. Because higher levels of APP have been correlated with Alzheimer's disease and Down syndrome, we asked whether levels of APL-1, the C. elegans APPrelated protein, have an effect on lifespan. Longevity in any organism is the result of a combination of environmental effects and genetic factors. C. elegans is constantly monitoring its surroundings and integrating this information to regulate its metabolic and reproductive processes, which ultimately determine the animal's lifespan. Several tissues, such as neurons, intestine, and gonad, have been implicated in regulating lifespan in C. elegans (Kenyon, 2010). For instance, ablation of certain amphidial sensory neurons can either lead to lifespan enhancement or reduction; similarly, ablation of germline precursor cells leads to an increased lifespan. These effects on lifespan are dependent in DAF-16 FOXO and DAF-12 VDR activity (Kenyon, 2010). The effects of APL-1 overexpression on lifespan were context dependent (Fig. 6e). Overexpressing apl-1 under its own promoter caused accelerated aging and lifespan reduction. By contrast, ubiquitous expression of apl-1 in adults caused lifespan extension. These results suggest that where APL-1 is expressed impacts its effects on lifespan. Because apl-1 expression in neurons, but not in hypodermal cells, is sufficient to rescue the lethality of apl-1 null mutants, we hypothesized that neuronal apl-1 expression was responsible for lifespan extension. Unexpectedly, the converse results were found; namely, apl-1 overexpression only in neurons led to lifespan reduction. However, apl-1 overexpression in hypodermal tissue is critical for lifespan extension and this lifespan extension is additive to the longevity induced by reduction in insulin signaling or by the removal of germline stem cells. Furthermore, we showed that expression of only the extracellular domain of APL-1 in these tissues was sufficient to cause these lifespan effects and lifespan extension is dependent on the activity of daf-16 FOXO and DAF-12 VDR. By contrast, hypodermal expression of apl-1 or APL-1EXT is not sufficient to rescue the lethality of apl-1 null mutants. Hence, the target of neuronal release of sAPL-1 vs. hypodermal release of sAPL-1 appears different. We hypothesize that migration of sAPL-1 is limited or confined by its local environment. For instance, sAPL-1 has two heparin binding domains (Fig. 4a), suggesting that the secreted APL-1 could interact locally with the extracellular matrix to limit its diffusion. Deleting the heparin binding domains of APL-1EXT resulted in a failure to rescue the apl-1 null lethality (unpublished observation). Furthermore, overexpression of APL-1EXT lacking the heparin binding domains had no effect on lifespan (Table S3), suggesting that interactions between sAPL-1 and the extracellular matrix are essential for APL-1 function. Collectively, these results indicate that APL-1 has multiple functions: expression of apl-1 in neurons is required for viability, whereas expression of apl-1 in hypodermal cells regulates longevity.
The lifespan effects due to apl-1 overexpression were not dependent on downregulating metabolic pathways via insulin/IGF-1 signaling or decreasing numbers of germline stem cells, but were influenced by the heterochronic pathway. apl-1 has been previously linked to the let-7 microRNA and heterochronic regulatory network (Niwa et al., 2008), which is composed of a regulatory cascade of protein regulators and microRNAs (miRNAs) that coordinate the onset of stereotypical stagespecific developmental events (Ambros & Horvitz, 1987;Rougvie & Moss, 2013). In particular, while levels of the LIN-14 transcription factor are negatively regulated by the lin-4 miRNA to allow transition from the first to the second larval stage (Lee et al., 1993), levels of lin-4 miRNA decrease with age, allowing LIN-14 levels to increase, thereby limiting the lifespan of C. elegans (Ib añez-Ventoso & Driscoll, 2009). We propose that hypodermal apl-1 expression represses levels of LIN-14, resulting in an increased lifespan relative to wild-type animals (Fig. 6e).
In C. elegans, maintenance of somatic tissues is lost during the first day of adulthood (Labbadia & Morimoto, 2014). This is consistent with the disposable soma theory that predicts that resources are allocated from the somatic tissue to the germ line (Kirkwood & Holliday, 1979), which happens when animals become reproductive during the L4-toadult transition. Interestingly, using a transcriptional reporter of the endogenous promoter of apl-1 expressing GFP, apl-1 has been shown by Niwa and colleagues to be transiently expressed under the control of let-7 miRNA network during the L4-to-adult transition in the hypodermal seam cells (Niwa et al., 2008). However, this hypodermal APL-1 expression is lost in adulthood and we show here that ectopically expressing APL-1 during adulthood in the hypodermis leads to increased health-and lifespan. We speculate that reinforcing APL-1 expression in the hypodermis is a reprogramming of developmental networks via heterochronic LIN-14 to improve the maintenance of For (a-c) P-value was determined by log rank. Statistics and additional lifespan data are in Table S4. somatic tissues via DAF-16/FOXO and HSF-1 that leads to improved organismal lifespan. The implication of an APP-related protein functioning in a miRNA and heterochronic gene network to promote healthy aging might point toward a possible entry to use miRNAs as a therapeutic intervention to modulate age-related consequences of APP function.

RNA interference
RNAi clones were picked from the Ahringer or Vidal libraries. Cultures were grown overnight in LB with 12.5 lg/mL tetracycline and 100 lg/ mL ampicillin, diluted to an OD600 of 1, and induced with 1 mM IPTG. This culture was seeded onto NGM agar plates containing tetracycline, ampicillin, and additional IPTG. Empty vector (EV) plasmid pL4440 was used as control.

Lifespan assays
Because our laboratory uses a different growing media (MYOB) than other laboratories, which normally use (NGM), and wild-type shows a different mean lifespan of 14 days on MYOB vs. 21 days on NGM (Keightley & Caballero, 1997), we performed most experiments on both media as indicated (Tables S1-S4). Lifespan results were similar on MYOB or on NGM. Note that the wild-type N2 var. Bristol strain has been maintained for many generations in different laboratories and can have mean lifespans ranging from 12 to 17 days at 20°C (Gems & Riddle, 2000). N2 animals from another laboratory (kindly provided by Cathy Savage-Dunn) showed a similar lifespan as our N2 stock on MYOB plates in four independent trials (198/398 animals). To determine whether the laboratory N2 animals had a longer lifespan on NGM plates, N2 animals were grown on MYOB until the L4 stage and then transferred onto NGM plates for lifespan analysis; these animals showed a mean lifespan of 18.62 AE 0.47 days (60/111 animals). These results indicate that the N2 mean lifespan of 14 days reported here is due to the different composition of the MYOB plates compared to NGM plates. For all lifespan trials, NGM: L4 animals were picked onto fresh OP50 plates, and then day one adults were placed on either OP50 or RNAi plates containing 50 lM 5-fluoro-2 0 -deoxyuridine (FUdR), unless otherwise indicated, as described in Ewald et al. (2015). All lifespan trials on MYOB were performed without FUdR. All strains were grown for at least two generations at 20°C, except for daf-2(e1370) and glp-1(e2141) animals, which were raised at 15°C. All adult lifespans [except glp-1 (e2141)] were measured starting from the L4 stage at 20°C. Animals carrying the temperature-sensitive glp-1(e2141) allele and appropriate control animals were shifted from 15°C to 25°C at the L1 stage and lifespan assays were performed at 25°C.

Pharyngeal pumping assays
The pharyngeal pumping rate was determined by the number of pumps per 20 s; measurements were only taken when worms were on bacteria and pumped at a constant rate without any pauses as described (Ewald et al., 2012b).

Autofluorescence assays
L4 animals were picked and scored at appropriate times. Animals were placed on an agar pad containing a drop of 10 mM NaN 3 . Pictures were taken with a Hamamatsu ORCA-ER digital camera on a Zeiss Axioplan microscope. The area directly behind the last bulb of the pharynx was photographed using Openlab 3.1.3 software (Improvision, Coventry, United Kingdom) with the same settings for all animals and conditions. The autofluorescence intensities were derived by selecting a defined area of the worm in the pictures and computing the 'mean gray value' by ImageJ software (NIH).

Western blot analysis
Preparations of animal lysates and Western blots were performed as described (Hornsten et al., 2007). Protein levels were normalized to levels of actin (~40 kDa) for each strain, so that similar amounts of total protein were loaded for each strain. For each blot, the blot was first probed with an actin monoclonal antibody (JLA20 at 1:2000; Developmental Studies Hybridoma Bank, Iowa City, Iowa, United States; secondary antibody anti-mouse-goat-HRP at 1:1000), washed three times with TAE, and then re-probed with an anti-APL-1 antiserum against the extracellular domain of APL-1 (Hornsten et al., 2007). Relative protein levels were determined by relative intensity to wildtype (N2) using NIH Image J Gel analyzer.

Quantitative real-time polymerase chain reaction (qRT-PCR) assays
Mixed-stage animals were harvested (>1000 animals; three independent trials; Figs. 5e-f and S3b,c,f). For adulthood qRT-PCR: one-day adults were placed on OP50 NGM FUdR plates at 20°C and 8 days later, 200 worms were harvested (Fig. 6d). RNA was isolated with Trizol (TRI REAGENT; Sigma, St. Louis, Missouri, United States), DNase-treated, and cleaned over a column (RNA Clean & Concentrator TM ; ZYMO Research, Irvine, California, United States). First-strand cDNA was synthesized in duplicate from each sample (Invitrogen, Carlsbad, California, United States, SuperScript III). SYBR green was used to perform qRT-PCR (Applied Biosystems 7900, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). For each primer set, a standard curve from genomic DNA accompanied the duplicate cDNA samples (Ewald et al., 2015). mRNA levels relative to N2 control were determined by normalizing to the number of worms and the geometric mean of three reference genes [cdc-42, pmp-3, and Y45F10D.4 (Hoogewijs et al., 2008)]. At least two biological replicates were examined for each sample. For statistical analysis, one-sample t-test, two-tailed, hypothetical mean of 1 was used for comparison using Prism 4.0a software (GraphPad, La Jolla, California, United States).

Fat quantification
Fixed Oil Red O (ORO) staining: Worms were synchronized by hypochlorite treatment and day 1 adult worms were fixed. ORO staining of fixed animals and quantification was performed.
Triglyceride quantification: Mixed-stage worms were collected and prepared as described. Triglyceride levels were measured with the Triglyceride Colorimetric Assay Kit (#10010303; Cayman Chemical, Ann Arbor, MI, USA) and normalized to protein levels using BCA protein assay (Pierce Biotechnology, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).

Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article:   Table S1 Hypodermal APL-1 increases lifespan. Table S2 APL-1 mediated lifespan extension requires DAF-16 and DAF-12. Table S3 APL-1 longevity requires functional APL-1 and is additive to reduced Insulin/IGF-1 and reduced germstem cell number mediated longevity.