The neuronal receptor tyrosine kinase Alk is a target for longevity

Abstract

Inhibition of signalling through several receptor tyrosine kinases (RTKs), including the insulin-like growth factor receptor and its orthologues, extends healthy lifespan in organisms from diverse evolutionary taxa. This raises the possibility that other RTKs, including those already well studied for their roles in cancer and developmental biology, could be promising targets for extending healthy lifespan. Here, we focus on anaplastic lymphoma kinase (Alk), an RTK with established roles in nervous system development and in multiple cancers, but whose effects on aging remain unclear. We find that several means of reducing Alk signalling, including mutation of its ligand jelly belly (jeb), RNAi knock-down of Alk, or expression of dominant-negative Alk in adult neurons, can extend healthy lifespan in female, but not male, Drosophila. Moreover, reduced Alk signalling preserves neuromuscular function with age, promotes resistance to starvation and xenobiotic stress, and improves night sleep consolidation. We find further that inhibition of Alk signalling in adult neurons modulates the expression of several insulin-like peptides, providing a potential mechanistic link between neuronal Alk signalling and organism-wide insulin-like signalling. Finally, we show that TAE-684, a small molecule inhibitor of Alk, can extend healthy lifespan in Drosophila, suggesting that the repurposing of Alk inhibitors may be a promising direction for strategies to promote healthy aging.

Keywords: Drosophila; Alk; aging; insulin/IGF signalling; lifespan; nervous system; receptor tyrosine kinase.

Figure 1

Heterozygous loss of jeb extends lifespan. (a) The original stock (BL10576) containing the jeb^k05644 mutation contained two insertions of P{lacW} that became apparent upon back‐crossing. Primers designed as shown amplified either P{lacW} independent of its insertion site (Set 1) or P{lacW} in the annotated insertion site in the jeb gene (Set 2). PCR from genomic DNA using these primers confirmed the presence of the jeb^k05644 insertion or the secondary extra insertion in each back‐crossed stock. (b) qPCR from whole‐fly RNA showed reduced jeb mRNA levels in female w^Dah;jeb^k05644/+; flies compared to w^Dah;+/+; flies, with increased jeb mRNA levels in w^Dah;extra‐insertion/+; flies compared to w^Dah;+/+; flies. n = 6 biological replicates of three whole flies per replicate for each genotype; p values are from Dunnett's multiple comparison tests between the indicated groups. (c) Survival curves showed extended lifespan for female w^Dah;jeb^k05644/+; flies compared to their w^Dah;+/+; siblings. (d) Survival curves showed no significant change in lifespan for female w^Dah;extra‐insertion/+; flies compared to their w^Dah;+/+; siblings. For all survival experiments, n > 145 deaths counted per condition; p values are from log‐rank tests vs. the wild‐type condition

Figure 2

RNAi knock‐down of Alk ubiquitously or in neurons extends lifespan. (a) mRNA expression data from FlyAtlas showed highly enriched expression of Alk mRNA in nervous system tissues (brain, head, eye and thoracic‐abdominal ganglion) compared to other adult tissues. (b, c) qPCR from head RNA showed reduced Alk mRNA levels in (b) w^Dah,UAS‐Alk^RNAi/w^Dah;Act‐GS/+; and (c) w^Dah,UAS‐Alk^RNAi/w^Dah;;elav‐GS^Tricoire/+ flies given food containing the inducing drug RU‐486 (200 μM) from 2 to 10 days of age compared to sibling flies of the same genotype treated with vehicle control food. n = 4 biological replicates of 6–7 heads per replicate for each condition; p values are from unpaired t tests between groups. (d) Survival curves showed extended lifespan for female w^Dah,UAS‐Alk^RNAi/w^Dah;Act‐GS/+ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. (e) Survival curves showed extended lifespan for female w^Dah,UAS‐Alk^RNAi/w^Dah;;elav‐GS^Tricoire/+ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. For all survival experiments, n > 160 deaths counted per condition; p values are from log‐rank tests vs. the vehicle‐treated (uninduced) group

Figure 3

Expression of dominant‐negative Alk in neurons extends lifespan. (a, b) qPCR from head RNA showed increased mRNA levels for (a) the extracellular region of Alk but not for (b) the intracellular region of Alk in female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ given food containing the inducing drug RU‐486 (200 μM) from 2 to 10 days of age compared to sibling flies of the same genotype treated with vehicle control food. n = 4 biological replicates of 6–7 heads per replicate for each condition; p values are from unpaired t tests between groups. (c) Survival curves showed no significant change in lifespan for female w^Dah;;elav‐GS³⁰¹/+ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. (d) Survival curves showed extended lifespan for female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. For all survival experiments, n > 140 deaths counted per condition; p values are from log‐rank tests vs. the vehicle‐treated (uninduced) group

Figure 4

Expression of dominant‐negative Alk in neurons improves climbing ability, resistance to starvation, and resistance to the xenobiotic toxin DDT. (a, b) The slope of age‐related decline in climbing ability was (a) not significantly changed for female w^Dah;;elav‐ GS³⁰¹/+ flies and (b) significantly improved for female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. Values shown are mean ± SEM from n > 45 individual flies for each condition and age; p values are from slope comparisons in a linear regression analysis. (c, d) Survival curves in starvation experiments started at 14 days of age showed (c) no significant change in survival for female w^Dah;;elav‐GS³⁰¹/+ flies and (d) significantly improved survival for female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. (e, f) Survival curves in DDT (0.03%) experiments started at 14 days of age showed (c) significantly decreased survival for female w^Dah;;elav‐GS³⁰¹/+ flies and (d) significantly improved survival for female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. For all survival experiments, n > 140 deaths counted per condition; p values are from log‐rank tests vs. the vehicle‐treated (uninduced) group

Figure 5

Expression of dominant‐negative Alk in neurons decreases night activity, increases night sleep and improves night sleep consolidation. (a) Activity traces and (b) quantification of day and night activity counts from mated female flies at 15 days of age showed significantly decreased night activity for w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. (c) Sleep traces, (d) quantification of day and night sleep, and (e) quantification of the number of day and night sleep bouts from mated female flies at 15 days of age showed significantly increased night sleep and significantly decreased numbers of night sleep bouts for w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies treated with RU‐486 (200 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. n = 32 individual flies per condition; p values are from Mann–Whitney tests vs. the vehicle‐treated (uninduced) group

Figure 6

Expression of dominant‐negative Alk in neurons reduces Erk phosphorylation and reduces mRNA levels for dilp2 and dilp5. (a, b) Western blots from head extracts showed decreased levels of phosphorylated Erk and no significant change in total Erk in female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies given food containing the inducing drug RU‐486 (200 μM) from 2 to 10 days of age compared to sibling flies of the same genotype treated with vehicle control food. n = 4 biological replicates of 12–15 heads per replicate for each condition; p values are from unpaired t tests between groups. (c) qPCR from head RNA showed decreased mRNA levels for dilp2 and dilp5, and no significant change in dilp6, in female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies given food containing the inducing drug RU‐486 (200 μM) from 2 to 10 days of age compared to sibling flies of the same genotype treated with vehicle control food. n = 4–5 biological replicates of 6–7 heads per replicate for each condition; p values are from unpaired t tests between groups. (d) qPCR from abdominal fat body RNA showed increased mRNA levels for dilp6 in female w^Dah;;UAS‐Alk^DN/elav‐GS³⁰¹ flies given food containing the inducing drug RU‐486 (200 μM) from 2 to 18 days of age compared to sibling flies of the same genotype treated with vehicle control food. n = 8 biological replicates of four abdominal carcasses per replicate for each condition; p value is from unpaired t test between groups

Figure 7

The Alk inhibitor TAE‐684, but neither alectinib nor crizotinib, extends lifespan in Drosophila. (a, b) Survival curves showed no significant change in lifespan for female w^Dah flies treated with Alectinib (10 μM) or Crizotinib (10 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. (c) Survival curves showed extended lifespan for female w^Dah flies treated with TAE‐684 (10 μM) from 2 days of age compared to sibling flies of the same genotype treated with vehicle control food. For all survival experiments, n > 135 deaths counted per condition; p values are from log‐rank tests vs. the vehicle‐treated group. (d, e, f) Diagrams show the amino acid residues within 3.5 A of each small molecule's binding site in published crystal structures with human Alk. Amino acids conserved between human and Drosophila are marked in bold; those not conserved show the Drosophila amino acid in parentheses