Cell type-specific modulation of healthspan by Forkhead family transcription factors in the nervous system

Abstract

Reduced activity of insulin/insulin-like growth factor signaling (IIS) increases healthy lifespan among diverse animal species. Downstream of IIS, multiple evolutionarily conserved transcription factors (TFs) are required; however, distinct TFs are likely responsible for these effects in different tissues. Here we have asked which TFs can extend healthy lifespan within distinct cell types of the adult nervous system in Drosophila Starting from published single-cell transcriptomic data, we report that forkhead (FKH) is endogenously expressed in neurons, whereas forkhead-box-O (FOXO) is expressed in glial cells. Accordingly, we find that neuronal FKH and glial FOXO exert independent prolongevity effects. We have further explored the role of neuronal FKH in a model of Alzheimer's disease-associated neuronal dysfunction, where we find that increased neuronal FKH preserves behavioral function and reduces ubiquitinated protein aggregation. Finally, using transcriptomic profiling, we identify Atg17, a member of the Atg1 autophagy initiation family, as one FKH-dependent target whose neuronal overexpression is sufficient to extend healthy lifespan. Taken together, our results underscore the importance of cell type-specific mapping of TF activity to preserve healthy function with age.

Keywords: Alzheimer’s disease; aging; glia; neurons; transcription factors.

Fig. 1.

Overexpression of foxo in glia, but not neurons, extends lifespan. (A) Images from the SCope database (33) show mRNA expression of foxo largely in repo-expressing (glial) cell populations in the Drosophila brain. (B) Immunofluorescence images from the cell body layer adjacent to the olfactory bulb in the central brain show FOXO-GFP expression overlapping with REPO-positive glial cells in w^Dah;foxo-GFP flies. (Scale bar, 10 μm.) (C and D) Survival curves show (C) shortened lifespan for w^Dah;;elav-GS/UAS-foxo flies and (D) extended lifespan for w^Dah;;GSG3285-1/UAS-foxo flies treated with 200 μM RU-486 from 2 d of age compared with sibling flies of the same genotype treated with vehicle control food. For all survival experiments, n > 140 deaths were counted per condition; P values are from log-rank tests between groups.

Fig. 2.

Overexpression of fkh in neurons, but not glia, extends lifespan independent of FOXO. (A) Images from the SCope database (33) show mRNA expression of fkh largely in nSyb-expressing (neuronal) cell populations in the Drosophila brain. (B) Immunofluorescence images from the cell body layer of the central brain show FKH expression in REPO-negative (neuronal) cells in w^Dah flies. (Scale bar, 10 μm.) (C–E) Survival curves show (C) extended lifespan for w^Dah;UAS-fkh/+;elav-GS/+ flies, (D) no change in lifespan for w^Dah;UAS-fkh/+;GSG3285-1/+ flies, and (E) extended lifespan for both w^Dah;UAS-fkh/+;elav-GS/+ and w^Dah;UAS-fkh/+;elav-GS, foxoΔ/foxoΔ flies treated with 200 μM RU-486 from 2 d of age, with no significant interaction between fkh overexpression and foxoΔ genotype. For all survival experiments, n > 85 deaths were counted per condition; P values are from either log-rank tests between groups (C and D) or Cox proportional hazards testing (E).

Fig. 3.

Overexpression of fkh in neurons improves neuromuscular function and reduces protein aggregation in the presence of Aβ. (A and B) Climbing assay results show (A) unaffected neuromuscular function in uninduced (vehicle control) w¹¹¹⁸;UAS-Aβ_Arc/UAS-fkh;elav-GS/+ flies compared with uninduced w¹¹¹⁸;UAS-Aβ_Arc/+;elav-GS/+ flies and (B) improved function in induced (200 μM RU-486) w¹¹¹⁸;UAS-Aβ_Arc/UAS-fkh;elav-GS/+ flies compared with induced w¹¹¹⁸;UAS-Aβ_Arc/+;elav-GS/+ flies. (C) Survival curves show no change in lifespan for induced (200 μM RU-486) w¹¹¹⁸;UAS-Aβ_Arc/UAS-fkh;elav-GS/+ flies compared with induced w¹¹¹⁸;UAS-Aβ_Arc/+;elav-GS/+ flies. (D and E) Immunostaining images (D) and quantification (E) show reduced accumulation of REF(2)P and polyubiquitinated protein (FK2) aggregates in induced w¹¹¹⁸;UAS-Aβ_Arc/UAS-fkh;elav-GS/+ flies compared with induced w¹¹¹⁸;UAS-Aβ_Arc/+;elav-GS/+ flies at 28 d of age. For climbing experiments, n = 3 vials of 15 flies per vial per condition; data are displayed as mean ± SEM; for survival experiments, n > 135 deaths were counted per condition; and for immunofluorescence experiments, n = 4 or 5 brains for uninduced and 11 to 13 brains for induced conditions. P values are from two-way ANOVA (A and B), log-rank test (C), or Bonferroni multiple comparisons (E).

Fig. 4.

RNA-seq from head mRNA reveals fkh-dependent transcriptional responses to reduced IIS. (A) In head mRNA from flies treated with 200 μM RU-486 or vehicle control from 2 to 5 d of age, the overlap between the transcriptional response from reduced IIS compared with controls (daGS>UAS-InR^DN,UAS-GFP-RNAi induced versus daGS>UAS-InR^DN,UAS-GFP-RNAi uninduced) and compared with flies with reduced IIS and fkh knockdown (daGS>UAS-InR^DN,UAS-GFP-RNAi induced versus daGS>UAS-InR^DN,UAS-fkh-RNAi induced), revealed 99 shared up-regulated and 84 shared down-regulated genes, which represented a significant overlap between comparisons (P = 6.3 × 10⁻⁴⁵ by Fisher’s exact test). (B) Heatmap shows P values for the 20 genes most significantly up-regulated or down-regulated among the shared genes between comparisons.

Fig. 5.

Overexpression of fkh in neurons increases Atg17 expression, and overexpression of Atg17 in neurons extends lifespan. (A) qPCR from head RNA shows increased mRNA levels for Atg17 in w^Dah;UAS-fkh/+;elav-GS/+ flies given food containing 200 μM RU-486 from 2 to 5 d of age. n = 4 or 5 biological replicates of 30 heads per replicate for each condition; the P value is by unpaired t test. (B) Survival curves show extended lifespan for w^Dah;UAS-Atg17/+;elav-GS/+ flies treated with 200 μM RU-486 from 2 d of age. n > 135 deaths were counted per condition; the P value is by log-rank test.