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

Significance Aging is the main risk factor for the costliest diseases in today’s world. However, significant gaps remain in understanding how different cell types modulate this most common physiological process. Here, we use published single-cell gene expression data to map the prolongevity roles of two evolutionarily conserved Drosophila transcription factors, FKH and FOXO, onto either neuronal or glial cells. We then demonstrate that neuronal FKH preserves healthy function even under stress. Finally, we identify an autophagy-related gene as one of FKH’s downstream prolongevity effectors. Our results exemplify tapping into publicly available gene expression datasets to extract physiological insights, and highlight the need to shift away from organism-wide approaches and toward cell type-specific strategies to obtain meaningful insights in aging research.

For images of FOXO, FKH, and REPO localization, image stacks were obtained on a Zeiss LSM700 confocal microscope using a 63x objective. For quantification of REF (2)P and ubiquitin (FK2) staining, image stacks of specimens were obtained using a 20× objective for imaging of the entire central brain. Stacks of 3 μm Z-steps and 6 images per stack were taken to capture the full depth of the cell body layers dorsal, ventral, and lateral to the antennal lobes. Images were quantified by an experimenter blinded to genotype using ImageJ software. Briefly, confocal stacks were merged into a single plane, then central brain region of each brain was manually traced using the DAPI channel. Thresholds were then set for REF (2)P and ubiquitin (with the same threshold used for all images), and the area above threshold within the region of interest measured.

RNA-Seq sample and library preparation.
Total RNA from fly heads (30 heads per sample) was extracted using the Qiagen total RNA isolation kit and quantified on an Agilent 2100 Bioanalyzer. Sample concentration and purity of RNA was measured on a NanoDrop spectrophotometer, and RNA integrity was assessed on an Agilent 2100 Bioanalyzer. Samples were processed using Illumina's TruSeq Stranded mRNA LT sample preparation kit (p/n RS-122-2101) according to manufacturer's instructions. Deviations from the protocol were as follows: 250ng total RNA was used as starting material; fragmentation was carried out for 10 minutes; and 14 cycles of PCR were used.
Briefly, mRNA was isolated from total RNA using Oligo dT beads to pull down poly-adenylated transcripts. The purified mRNA was fragmented using chemical fragmentation (heat and divalent metal cation) and primed with random hexamers. Strand-specific first strand cDNA was generated using Reverse Transcriptase and Actinomycin D to allow for RNA-dependent synthesis while preventing spurious DNAdependent synthesis. The second cDNA strand was synthesized using dUTP in place of dTTP, to mark the second strand.
The resultant cDNA was then "A-tailed" at the 3' end to prevent self-ligation and adapter dimerization. Full length TruSeq adaptors, containing a T overhang, were ligated to the A-Tailed cDNA. These adaptors contained sequences that allow the libraries to be uniquely identified by way of a 6bp Index sequence. Successfully ligated fragments were enriched with 14 cycles of PCR. The polymerase used was unable to read through uracil, so only the first strand was amplified, thus making the library strand-specific.
Sequencing. Libraries to be multiplexed in the same run were pooled in equimolar quantities, calculated from Qubit and Bioanalyzer fragment analysis. Samples were sequenced on the NextSeq 500 instrument (Illumina, San Diego, US) using a 43bp paired end run resulting in >15million reads per sample. Sequencing was carried out by UCL Genomics at the UCL GOS Institute of Child Health.

Quantitative real-time PCR (qPCR).
Total RNA was isolated from fly heads using standard Trizol (Invitrogen) protocols. RNA samples were treated with Turbo DNase (Invitrogen) and converted to cDNA using oligod(T) primers and Superscript II reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (ABI) in the Quant Studio 6 Flex system. Relative quantities of transcripts were determined using the relative standard curve method normalized to Tub84B. Primer sequences were: Atg17_F: GGGCTCCAAGTTCTATCGCA; Atg17_R: CTGATAGACGCTCGTGTTGC; Tub84B_F: TGGGCCCGTCTGGACCACAA; Tub84B_R: TCGCCGTCACCGGAGTCCAT.
Western blots. For total protein extraction (ATG8 western blots), 8 fly heads per sample were homogenized in 1X RIPA buffer (NEB 9806) with protease inhibitors (Roche, Cat# 11 836 170 001), and centrifuged for 2 min at 13,000 rpm in 40C. The resulting supernatant fraction was collected for western blot. Equal quantities of protein for each sample (as determined by the BCA Protein Assay Kit (Pierce)) were then separated on Poly-Acrylamide gels (15%, Acrylamide/Bis-Acrylamide solution, Sigma A7168, made according to manufacturers recommended protocol) and transferred to a PVDF membrane.
For insoluble ubiquitinated proteins, western blots were carried out as described in detail in (18). Briefly, 10 fly heads per sample were homogenized in Triton-X buffer (1% Triton-X, 10mM NEM, 50μm MG132, Complete Mini protease inhibitors (Roche), in PBS). After centrifugation, the supernatant was collected as the soluble fraction and used for protein quantification and loading control (Actin) gels. The insoluble pellet was re-suspended in SDS buffer (2% SDS, 10mM NEM, 50μm MG132, Complete Mini protease inhibitors (Roche), 50mM Tris pH 7.4), centrifuged, and the supernatant collected as the insoluble sample. Equal quantities of protein for each sample were then separated on 4-12% NuPage Bis-Tris gels (Invitrogen) and transferred to PVDF membranes.