The calcineurin inhibitor Sarah (Nebula) exacerbates Aβ42 phenotypes in a Drosophila model of Alzheimer's disease

Abstract

Expression of the Down syndrome critical region 1 (DSCR1) protein, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, is elevated in the brains of individuals with Down syndrome (DS) or Alzheimer's disease (AD). Although increased levels of DSCR1 were often observed to be deleterious to neuronal health, its beneficial effects against AD neuropathology have also been reported, and the roles of DSCR1 on the pathogenesis of AD remain controversial. Here, we investigated the role of sarah (sra; also known as nebula), a Drosophila DSCR1 ortholog, in amyloid-β42 (Aβ42)-induced neurological phenotypes in Drosophila. We detected sra expression in the mushroom bodies of the fly brain, which are a center for learning and memory in flies. Moreover, similar to humans with AD, Aβ42-expressing flies showed increased Sra levels in the brain, demonstrating that the expression pattern of DSCR1 with regard to AD pathogenesis is conserved in Drosophila. Interestingly, overexpression of sra using the UAS-GAL4 system exacerbated the rough-eye phenotype, decreased survival rates and increased neuronal cell death in Aβ42-expressing flies, without modulating Aβ42 expression. Moreover, neuronal overexpression of sra in combination with Aβ42 dramatically reduced both locomotor activity and the adult lifespan of flies, whereas flies with overexpression of sra alone showed normal climbing ability, albeit with a slightly reduced lifespan. Similarly, treatment with chemical inhibitors of calcineurin, such as FK506 and cyclosporin A, or knockdown of calcineurin expression by RNA interference (RNAi), exacerbated the Aβ42-induced rough-eye phenotype. Furthermore, sra-overexpressing flies displayed significantly decreased mitochondrial DNA and ATP levels, as well as increased susceptibility to oxidative stress compared to that of control flies. Taken together, our results demonstrating that sra overexpression augments Aβ42 cytotoxicity in Drosophila suggest that DSCR1 upregulation or calcineurin downregulation in the brain might exacerbate Aβ42-associated neuropathogenesis in AD or DS.

Keywords: Alzheimer's disease; Amyloid-β42; DSCR1 (RCAN1); Drosophila; sarah (nebula).

Fig. 1.

Intrinsic sra expression is shown in the mushroom bodies and photoreceptor neurons, and is upregulated by Aβ42 expression. (A-D) Expression patterns of sra were examined using enhanced green fluorescence protein (EGFP) activity and anti-Sra antibody staining in Drosophila mushroom bodies (A,A″,B,B′; ×200) and third-instar larval eye imaginal discs (C,C″,D,D″; ×1200). Mushroom bodies and photoreceptor neurons are highlighted by staining with anti-Fas-II (A′,A″; ×200) and anti-Chaoptin (C′,C″,D′,D″; ×1200, 24B10) antibodies, respectively. (E,F) Sra mRNA (E) and protein (F) levels were upregulated in Aβ42-expressing flies (elav>Aβ42) compared with those of the control (elav-GAL4 or UAS-Aβ42). All data are expressed as mean±s.e.m. (E, Tukey–Kramer test, n≥6, ***P<0.001; F, Tukey–Kramer test, n=10, **P<0.01, ***P<0.001). Fas-II, Fasciclin II.

Fig. 2.

Overexpression of sra exacerbates Aβ42-induced phenotypes in Aβ42-expressing flies. (A-M) The eye phenotypes induced by ectopic expression of Aβ42 in the developing eye were aggravated by sra overexpression. The Aβ42-expressing adult eye (H) was severely distorted as a result of neurodegeneration when compared with the control eye (A,B,G). Overexpression of sra alone resulted in a marginally rough-eye phenotype (C,D) compared with that of the control (A,B,G). Overexpression of sra in Aβ42-expressing flies exacerbated the rough-eye phenotype (H-J). By contrast, sra deficiency (E) partially rescued the rough-eye phenotype (K) as compared with that of Aβ42-expressing flies (H). The rough-eye phenotype induced by sra overexpression was rescued by DIAP1 overexpression (F,L). Inset figures are high-magnification images. (M) The graph shows the relative eye size of each experimental group (Tukey–Kramer test, n≥9, *P<0.05, ***P<0.001). (N-P) Aβ42 levels do not change following expression of sra. (N,O) Aβ42 mRNA (N) and protein (O) in the larval eye discs of each group (N, Student's t-test, n=8; O, Student's t-test, n=7; NS, not significant). (P) Confocal images showing the presence of Aβ42 in larval eye discs of the indicated groups. More than 20 discs were observed for each group, and the representative images are shown. Magnification of the pictures, ×200. (Q) Representative images of Thioflavin-S staining in the brains of 20-day-old male flies. No prominent difference in staining was observed between brains of homozygous Aβ42-expressing flies with (elav>Aβ42+sra^EY) or without (elav>Aβ42) sra overexpression. No signal was detected in the control (elav-GAL4). Magnification of the pictures, ×400. (R) Survival rates of pan-neuronal Aβ42-expressing flies with sra overexpression (elav>Aβ42+sra^EY) during development. The effects of overexpressed sra (elav>sra^EY) in the controls (elav-GAL4, UAS-Aβ42, sra^EY) are also shown (Tukey–Kramer test, n≥250, *P<0.05, **P<0.01, ***P<0.001). (S) Effect of sra overexpression on the locomotor activity of pan-neuronal Aβ42-expressing flies. Climbing assay was performed using 10-day-old male flies (Tukey–Kramer test, n=100, ***P<0.001, NS, not significant). (T) Survival curve of pan-neuronal Aβ42-expressing male flies with sra overexpression (elav>Aβ42+sra^EY). The lifespans of sra- (elav>sra^EY) or Aβ42-(elav>Aβ42) expressing flies and control flies (elav-GAL4) are also presented (Kaplan–Meier estimator and log-rank test, n≥100). All data are expressed as mean±s.e.m.

Fig. 3.

Overexpression of sra induces neurodegeneration and aggravates Aβ42-induced phenotypes in Aβ42-expressing flies. (A) Acridine orange (AO)-staining images of larval brains. (B) The graph shows the average number of AO-positive cells in the larval brains of each experimental group (Tukey–Kramer test, n=10, ***P<0.001). The data are expressed as mean±s.e.m. (C) Overexpression of sra increased Aβ42-induced defects in photoreceptor axon targeting. Photoreceptor axon projections in a late third-instar larval brain were stained with an anti-Chaoptin antibody (24B10). The anti-Elav antibody highlights whole neurons. Magnification of the pictures, ×400.

Fig. 4.

Pan-neuronal overexpression of sra increases the number of glial cells and nitric oxide (NO) levels in Drosophila brains. (A) Representative immunohistochemistry images of larval brain stained with an anti-Repo antibody. (B) Confocal images of the larval brains with indicated genotype corresponding to the white box in A. Magnification of the pictures: (A) ×200, (B) ×400. (C) The graph shows the number of Repo-positive cells (Tukey–Kramer test, n≥20, **P<0.01, ***P<0.001, NS, not significant). (D) Overexpression of sra or Aβ42 increases NO levels in the adult fly head region (Tukey–Kramer test, n≥6, ***P<0.001, NS, not significant). All data are expressed as mean±s.e.m.

Fig. 5.

Overexpression of sra increases oxidative stress susceptibility and induces mitochondrial dysfunction. (A) Survival rates of Aβ42-expressing flies overexpressing sra under oxidative stress conditions (n=200). The Kaplan–Meier estimator and log-rank test was used to determine significant differences in survival rates of samples. elav-GAL4 vs elav>sra^EY: P=0.0001; elav-GAL4 vs elav>Aβ42: P<0.0001; elav>sra^EY vs elav>Aβ42+sra^EY: P=0.0001; elav>Aβ42 vs elav>Aβ42+sra^EY: P=0.0004. (B) The relative levels of mtDNA were determined with real-time quantitative PCR using primers for mitochondrial cytochrome b (Cyt b) and mitochondrial cytochrome c oxidase subunits I and III (Co I and Co III). Tukey–Kramer test, n=9, *P<0.05, **P<0.01, ***P<0.001, NS, not significant. (C) Overexpression of sra decreased ATP levels in Aβ42-expressing flies. ATP levels were measured using a bioluminescent assay (Tukey–Kramer test, n≥4, *P<0.05, **P<0.05, ***P<0.001). (D) Relative mRNA levels of ROS response-associated genes were determined with real-time quantitative PCR using primers for superoxide dismutase subunits 1, 2 and 3 (SOD1, SOD2 and SOD3) and glutathione S transferase D1 (GstD1) (Tukey–Kramer test, n≥18, *P<0.05, **P<0.01, ***P<0.001, NS, not significant). All data are expressed as mean±s.e.m.

Fig. 6.

The phenotypes of Aβ42-expressing flies are exacerbated by chemical calcineurin inhibitors or calcineurin RNAi. (A-C,F-H) Representative images of developing eye phenotypes in DMSO-fed (A,F), FK506-fed (B,G) and CsA-fed (C,H) flies with (F,G,H) or without (A-C) Aβ42 expression. (D,E,I,J) Representative images of developing eye phenotypes in flies expressing calcineurin A1 RNAi (CanA1i) or calcineurin B RNAi (CanBi) with (I,J) or without (D,E) Aβ42. Inset figures are high-magnification images. (K) The graph shows the relative eye size in each experimental group (Tukey–Kramer test, n≥10, *P<0.05, ***P<0.001). (L) The effect of CanA1 knockdown on the survival rate of Aβ42-expressing flies (Tukey–Kramer test, n≥350, ***P<0.001). (M) CanA1 knockdown in neurons increased the number of glial cells in the larval brain. The graph shows the number of Repo-positive cells (Tukey–Kramer test, n≥15, ***P<0.001, NS, not significant). CsA, cyclosporin A.