Amyloid-beta (Aβ1–42)-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply

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Amyloid-beta (A␤1–42 )-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply

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Charlotte Regitz, Uwe Wenzel ∗

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Q2 Molecular Nutrition Research, Interdisciplinary Research Center, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany

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h i g h l i g h t s • • • •

Cholesterol restriction decreases A␤1–42 -induced toxicity in C. elegans. The apolipoprotein homologue vitellogenin-6 mediates cholesterol uptake. Cholesterol triggers the steroidal-signaling pathway. Interactions between steroidal and insulin/IGF signaling affect A␤1–42 -toxicity.

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Article history: Received 28 January 2014 Received in revised form 5 May 2014 Accepted 29 May 2014 Available online xxx

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Keywords: Caenorhabditis elegans Amyloid ␤ peptide Alzheimer’s disease Vitellogenin Cholesterol

Alzheimer’ disease is a neurodegenerative disorder characterized by the misfolding and aggregation of amyloid ␤ (A␤). This process is influenced through supply of cholesterol via apolipoproteins to neurons. In the present study, we used the transgenic Caenorhabditis elegans strain CL2006, which expresses A␤1–42 under control of a muscle-specific promoter, to test the effects of the apolipoprotein B homologue vitellogenin-6 on paralysis. Knockdown of vitellogenin-6 using RNA-interference (RNAi) recently was shown to significantly reduce cholesterol absorption in C. elegans, and both, RNAi for vitellogenin-6 or lowering the cholesterol concentration in the medium was associated with reduced A␤-aggregation and paralysis in the nematodes. The effects of both interventions are mediated through the inhibition of the steroidal signaling pathway since knockdown of its key factors DAF-9 or DAF-12 reduced paralysis independent of the cholesterol concentration and without additive effects by vitellogenin-6 RNAi. Double-RNAi for daf-12 and the downstream target of insulin signaling, the foxo transcription factor daf16, revealed that the paralysis-triggering effects of daf-16 RNAi were dominant over the preventive effects of daf-12 RNAi. Identical observations were made when the transcriptional co-activators of DAF-16, ftt-2 or par-5 were knocked down instead of daf-16. In conclusion, interactions between the steroidal and insulin-signaling pathways were identified in A␤1–42 expressing CL2006, where cholesterol deprivation inhibits steroidal signaling and thereby activates insulin signaling. Those effects were associated with a reduced Alzheimer phenotype in the nematodes, i.e. reduced protein aggregation and paralysis. © 2014 Published by Elsevier Ireland Ltd.

Alzheimer’ disease (AD) is a neurodegenerative disorder characterized by the misfolding and aggregation of amyloid ␤ (A␤) into fibrils which can confer profound cytotoxicity leading to the progressive impairment in memory and cognition [31,32]. Plasma cholesterol levels represent a factor often linked to AD although it must be emphasized that study results are rather inconsistent as long where long follow-ups and the effects of midlife lipid

Abbreviations: A␤, amyloid beta peptide; AD, Alzheimer’s disease; IGF, insulinlike growth factor; RNAi, RNA-interference. ∗ Corresponding author. Tel.: +49 641 99 39220; fax: +49 641 99 39229. E-mail address: [email protected] (U. Wenzel).

levels on late-life cognitive function remain unconsidered [34]. Apolipoprotein E (apoE) still represents the major susceptibility gene for AD, encoding a factor which contributes to the distribution of cholesterol from low-density lipoproteins to peripheral tissues but which is also essentially involved in the transport of lipids in the brain and cerebrospinal fluid [25,27]. In general, the ␧2 allele of apoE is associated with lower plasma cholesterol level and a reduced risk of AD. Whereas the ␧4 allele is linked to the opposite situation, and the more common ␧3 allele is associated with average plasma cholesterol and AD-risk [9,25,29,34]. Representing the major apolipoprotein in the central nervous system, ApoE could affect AD-development through many activities, such as promoting the formation of A␤ fibrils [14,33] or by influencing

http://dx.doi.org/10.1016/j.neulet.2014.05.059 0304-3940/© 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: C. Regitz, U. Wenzel, Amyloid-beta (A␤1–42 )-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.059

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Fig. 1. Paralysis (A) and aggregated protein in solution (B) is significantly increased in CL2006 containing the A␤1–42 transgene in comparison to N2 wildtype nematodes. Reduced cholesterol supply to CL2006 results in reduced paralysis (C) and reduced amounts of aggregated protein in solution (D). Paralysis was measured on agar plates in young adult nematodes subsequent to a heat shock for 1 h at 35 ◦ C in NGM. Nematodes, treated either with vit-6 RNAi, low cholesterol (2.5 ␮g/ml cholesterol, standard concentration was 5 ␮g/ml), or a combination of both, were classified as paralyzed when they responded to tapping with a bented platinum wire only with movement of the head but not with the body. The amount of aggregated protein in solution was measured by the use of ProteoStat® Protein aggregation assay which contains a fluorescent dye that binds specifically to protein aggregates **p < 0.01, ***p < 0.001versus the vector control.

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the intracellular degradation of A␤ as affected by intracellular cholesterol level [20,33]. Another gene implicated in AD which is associated with lipid metabolism is sorl-1, encoding the sortilinrelated receptor containing LDL-receptor class A repeats, which emerges as a key player in amyloidogenic processing [34,36]. Since the blood–brain-barrier prevents direct transport of cholesterol between the peripheral circulation and the brain, the identification of permeable 27-hydroxycholesterol, an oxysterol that impairs memory functions, could provide the missing link between AD and hypercholesterolemia [6,34]. In the present study we used the transgenic C. elegans strain CL2006, which expresses the human 42 amino acid sequence of A␤ under the control of the muscle-specific unc-54 promoter/enhancer of C. elegans [23] and responds to increased temperatures of 35 ◦ C for 1 h with paralysis in ca. 50% of the population. Paralysis was measured using visual analysis as described for a transgenic strain with temperature-dependent expression of A␤ that leads to movement in induced animals which is restricted to waving of the head, with no translocation of the animal [24]. It is important to note that the A␤ finally expressed is a 3–42 truncation product [28]. In vitro analysis demonstrates that A␤3–42 self-aggregates more rapidly than A␤1–42 , forms fibrillar structures, and potently initiates A␤1–42 aggregation [28]. Of the various A␤ forms that exist, which differ in their molecular size or with regard to truncation or pyroglutamylation [31], A␤3–42 seems to have the highest propensity to form highly toxic A␤ aggregates also in humans, especially when pyroglutamylated by glutaminyl cyclase posttranslational at its N-terminus [30]. Beyond feeding low cholesterol concentrations (2.5 ␮g/ml in NGM) to CL2006,

RNA-interference (RNAi) for vit-6, a factor needed for intestinal cholesterol absorption [10] was performed to assess the influence of cellular cholesterol supply on paralysis. RNAi was also accomplished to achieve knockdowns of daf-9 and daf-12, two key members of the steroidal-signaling pathway [11], and of daf-16, a key transcription factor regulated by insulin/insulin-like growth factor (IGF) signaling, which has been demonstrated to be very important for stress response and longevity in C. elegans [22] and also in mammals [2]. Relative mRNA levels in nematodes cultured on RNAi bacteria (L4440) versus the vector control were determined by quantitative RT-PCR and normalized to 18S rRNA. The expression of knocked down genes was below 0.25 for all genes tested versus the vector control (p < 0.01). Aggregated proteins in solution were determined using the ProteoStat® protein aggregation assay. All experiments including a final heat-shock at 35 ◦ C for 1 h were performed in liquid culture as described [10,18,35]. Subsequently, nematodes were transferred to agar plates by pipetting 50 individuals and paralysis was measured. For group comparisons, analysis of variance (ANOVA) was performed. Differences between groups were determined by Bonferroni–Holm multiple comparison test. For statistical analysis of differences between two groups a student’s t-test (GraphPadPrism 5.0, La Jolla, CA, USA) was used. All paralysis measurements were repeated thrice. Results are presented as the means ± SD with significance levels of p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), respectively. Temperatures of 35 ◦ C for 1 h turned out to cause paralysis in ca. 50% of the CL2006 nematodes (with A␤1–42 transgene), whereas no significant paralysis was present under these conditions in N2 wildtype nematodes (without A␤1–42 transgene) (Fig. 1A). Those

Please cite this article in press as: C. Regitz, U. Wenzel, Amyloid-beta (A␤1–42 )-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.059

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Fig. 2. The steroidal-signaling pathway triggers paralysis in CL2006. Nematodes were grown either in the presence of the E. coli HT115 carrying the vector plasmid or the plasmid with an integrated daf-9 (A) or daf-12 (B) sequence. Prior to feeding dsRNA synthesis was induced. Nematodes treated with vector controls or RNAi for daf-9 or daf-12 were tested for paralysis after they had been grown at normal (5 ␮g/ml cholesterol) or low cholesterol (2.5 ␮g/ml cholesterol) supply). In the vector control 53.3 ± 4.5% of the nematodes were paralyzed. ***p < 0.001 versus the vector control.

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effects were reflected at the level of aggregated proteins in solution which was increased in CL2006 8.4 fold over that of the wildtype nematodes (Fig. 1B). Both the knockdown of the apolipoprotein homologue vit-6 or the lowering of cholesterol provided by food reduced the paralysis (Fig. 1C) and also the amount of aggregated protein in solution (Fig. 1D). The finding that knockdown of vit6 under restricted cholesterol supply did not result in a stronger reduction of paralysis or aggregated protein than each of the interventions alone (Fig. 1C and D) suggests that both act via the same pathway on the AD-phenotype in CL2006. Since we have previously shown that VIT-6 increases the resistance versus the nematicidal bacterial pathogen Photorhabdus luminescens by providing cholesterol for cytochrome P450 dependent DAF-9, which then generates steroidal ligands, the dafachronic acids, for the nuclear hormone receptor DAF-12 [10], we were interested in the effects of this steroidal-signaling pathway on paralysis in CL2006. Indeed the knockdown of both key factors of steroidal-signaling in C. elegans reduced the paralysis rate in CL2006 significantly (Fig. 2). Moreover, neither RNAi for vit-6 or feeding the nematodes low cholesterol concentrations resulted in additional effects on the reduced paralysis which was due to daf-9 (Fig. 2A) or daf-12 (Fig. 2B) RNAi. In a previous study we found that an active steroidal-signaling pathway, supplied by vitellogenins with its substrate cholesterol, in the presence of pathogenic bacteria was associated with reduced nuclear translocation of the forkhead transcription factor DAF-16 [10]. Recent findings supports that steroidal-signaling can also be placed upstream of DAF-16 [26] whereas DAF-9 and DAF-12 especially with regard to larval development typically act downstream [1,15]. Besides interacting with steroidal-signaling, DAF-16 provides the major downstream effector of the insulin/IGF-signaling pathway which is negatively regulated through it [19]. Insulin/IGFsignaling is evolutionary highly conserved and via the regulation of DAF-16 it possesses great influence on development [13,16], longevity [17], metabolism [3,7], and stress resistance [4]. Moreover, the 14-3-3 proteins FTT-2 and PAR-5 were shown to be necessary for the transcriptional activation of DAF-16 target genes and the promoted stress resistance under distinct conditions [5,12]. Here we show, that knockdown of daf-16 and also of ftt-2 or par5 significantly increased paralysis in CL2006 (Fig. 3). All three knockdowns increased the paralysis rate in wildtype nematodes slightly but not significant (vector control: 3.6 ± 1.2%, daf-16 RNAi: 6.3 ± 2.8%, ftt-2 RNAi: 5.1 ± 2.0, par-5 RNAi: 7.0 ± 3.4; for all p > 0.05 versus the vector control). The effects of par-5 RNAi were not as

heavily expressed as those of the daf-16 or ftt-2 knockdown. This fits to findings showing that in contrast to ftt-2, RNAi knockdown of par-5 did not show notable effects on dauer formation, DAF-16 localization, or DAF-16 downstream gene transcription [21]. Importantly, RNAi for daf-16 or its transcriptional co-activators prevented

Fig. 3. The steroidal-signaling pathway interferes with insulin-signaling. RNAi for the forkhead transcription factor daf-16 increases paralysis in CL2006 significantly and prevents the paralysis reducing activity of daf-12 RNAi (A). Knockdown of ftt2 (B) and par-5 (C) also increases paralysis and acts epistatic over daf-12 RNAi effects (see Fig. 2 for effects of daf-12 RNAi). In the vector controls 42.3 ± 4.1% (A), 40.0 ± 4.0% (B), and 54.6 ± 5.3% (C) of the nematodes were paralyzed. *p < 0.05, **p < 0.01, ***p < 0.001 versus the vector control.

Please cite this article in press as: C. Regitz, U. Wenzel, Amyloid-beta (A␤1–42 )-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.059

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the paralysis-reducing activities of daf-12 RNAi (Fig. 3), indicating that DAF-16 acts downstream of the steroidal signaling pathway regarding its effects on A␤-exerted degeneration. Almost identical results were obtained when daf-9 was knocked down. These results finally demonstrate that DAF-12 in its liganded or unliganded form inhibits the transcriptional activity of DAF-16. Although DAF-12 has been shown in many studies to link developmental progression and longevity [1] it is important to note that it can also shorten life span depending on the insulin/IGF-signaling state and relative levels of DAF-16 activity [8]. In conclusion, our study shows that lowering the cholesterol supply reduces the A␤-induced degeneration in an Alzheimer model of the nematode C. elegans and, to our knowledge, shows for the first time that inhibition of the steroidal-signaling pathway, as achieved by lowering the cholesterol supply or functional loss of vit-6, inhibits the A␤-induced phenotype, i.e. paralysis and protein aggregation, through activation of the transcriptional activity of the forkhead transcription factor DAF-16. Acknowledgement

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We greatly acknowledge Mr. Alan McDonley for critical reading of the manuscript.

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