Neuronal expression in Drosophila of an evolutionarily conserved metallophosphodiesterase reveals pleiotropic roles in longevity and odorant response

Abstract

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.

Fig 1. dMpped is a metallophosphoesterase and its expression is enriched in neurons.

(A) Sequence alignment of dMpped with human MPPED1, human MPPED2 and rat MPPED2. Red boxes highlight the conserved residues characteristic of metallophosphoesterases. * indicates residues D49 and H52 mentioned in the text. Highlighted in the blue box is the histidine residue that distinguishes MPPED1 from MPPED2. (B) A Coomassie R stained gel showing purified dMpped protein used for biochemical assays. (C) The catalytic activity of dMpped with the indicated colorigenic substrates (10 mM), in the presence of Mn²⁺ (5 mM) with dMpped (500 ng protein). Values represent the mean ± S.E. of duplicate determinations of experiments performed using two independent protein preparations. BispNPP, bis(p-nitrophenyl) phosphate; pNPPP, p-nitrophenyl phenylphosphonate; pNPP, p-nitrophenyl phosphate; TmPP, thymidine 5’-monophosphate-p-nitrophenyl ester; pNPPC, p-nitrophenylphosphoryl-choline. (D) Expression of CG16717 (dMpped) obtained from an analysis of RNAseq data from the modEncode data hosted in Flybase (www.flybase.org). Transcript levels are seen at low levels in many tissues, including the CNS and larvae (E) Expression pattern of dMpped across developmental stages of the fly. Embryos (2 h after egg laying; 50), third instar larvae (wandering; 10), pupae (21 h after pupae formation; 10), male and female flies (3 days old; 10 each) were collected and RT-qPCR was performed. dMpped transcript levels have been normalized to RpL32 transcript levels. The graph represents mean from 2 sets of samples collected independently. (F) Expression pattern of dMpped in different adult tissues. Testis and ovaries were collected from 50 male and female flies, respectively. The intestine (gut) was dissected from ∼ 50 flies in total, and 100 flies were used for muscles and brains. The graph represents mean from 2 sets of samples collected independently. (G) Confocal images of the ovary, testis, posterior midgut (R3-R4) and brain in pBAC(IT.GAL4)CG16717/UAS-mCD8GFP. pBac(IT.GAL4) enhancer trap element is positioned within the CG16717 gene. Ovaries show expression in the oviduct, the testis in secondary cells, and scattered enterocytes are GFP-positive in the posterior midgut. In the brain, strong expression is seen in the antennal lobe, marked with white asterix and in the optic lobe. The scale bar indicates 100 μm. 3D renderings of the brain sections of female brains are shown in S1 Movie, and enlarged images are shown in S1 Fig.

Fig 2. RNA-seq analysis and misregulation of genes associated with defense pathways.

(A) A volcano plot showing protein-coding genes altered in 35-day old dMpped^KOflies. The Log₂ fold change (Log₂FC) values are plotted on the x-axis and the negative log (base 10) of the Q value (-Log₁₀Q_value) on the y-axis. Blue (upregulated) and red (downregulated) dots represent genes with Q-values <0.05 and Log2 fold change values of more or less than one-fold. (B) The tissue distribution of differentially regulated genes. Misregulated genes (852, with 592 up-regulated and 260 down-regulated) were analysed by DGET (http://www.flyrnai.org/tools/dget/web/) based on published analysis of the Drosophila transcriptome [88]. (C) KEGG analysis of genes with Log2FC >2. Apart from several genes of unknown function, many misregulated genes were associated with defense response and immunity. (D) dMpped^KO flies were crossed with Dredd hypomorph flies (P[39]DreddEP1412 w¹¹¹⁸) and life span monitored in virgin female flies. Flies (w¹¹¹⁸, n = 76; dMpped^KO n = 66; Dredd, n = 40; Dredd;dMpped^KO, n = 59) were from at least three independent experiments. Log-rank test was used to compare survival across genotypes. Values shown at each time point are the mean ± SD, and the line represents the average across all experiments. p values are shown and compare the average life span across all three replicates of each genotype. (E) RT-qPCR of AMPs from RNA prepared from whole flies. Reducing Dredd activity in dMpped^KO flies reduced expression the AMPs, indicating that dMpped^KO flies showed misregulation of the IMD pathway. Each data point represents RNA prepared from 10 female flies, and values shown are the mean ± SD of three independent experiments. Data were analyzed by ANOVA and corrected for multiple comparisons using Tukey’s test. p values are indicated across genotypes.

Fig 3. Neuronal expression of dMpped determines fly lifespan, AMP expression and cyclic nucleotide levels in the brain.

(A) Western blot to confirm that dMpped^KO is a protein null allele, and expression of dMpped in the brain of flies where dMpped expression is driven in the neurons. The blot was re-probed with an anti-tubulin antibody to normalize protein loading. (B) Survival curves of backcrossed flies of indicated genotypes. Flies (w¹¹¹⁸, n = 66; dMpped^KO, n = 66; elav-Gal4>dMpped; dMpped^KO, n = 30) were from at least three independent experiments. Log-rank test was used to compare across genotypes. Values shown at each time point are the mean ± SD, and the line represents the average across all experiments. p values are shown and compare the average life span across all three replicates of each genotype. (C) RT-qPCR to confirm upregulation of AMPs in dMpped^KO flies. Levels were restored following neuronal expression of dMpped in dMpped^KO flies. Each data point represents RNA prepared from 10 female flies, and histograms correspond to the mean value ± SD of three independent experiments. Data were analyzed by ANOVA and corrected for multiple comparisons using Tukey’s test. p values are indicated across genotypes. (D) Cyclic AMP and Cyclic GMP levels were measured in the heads of flies of the indicated genotypes. Levels of cAMP and cGMP were significantly higher in dMpped^KO flies, correlated with reduced phosphodiesterase activity in flies deleted for dMpped. Extracts were prepared from 10 female fly heads, and histograms correspond to the mean value ± SD of three independent experiments. Data were analyzed by ANOVA and corrected for multiple comparisons using Tukey’s test. p values are indicated across genotypes.

Fig 4. dMpped regulates expression of Obps and affects odorant-driven behavior and tolerance to desiccation.

(A) Expression of dMpped in specific neurons in the fly brain. Data was extracted from single cell RNAseq analysis [47] and shown as a heat map indicating expression levels in flies of 3 and 30 days of age. The highest expression is seen in OPNs in agreement with Fig 1G. (B) RT-qPCR of Obp28 and Obp59 indicating misregulation in dMpped^KO flies with levels restored to that seen in control flies following neuronal expression of dMpped in dMpped^KO flies. At least 10 flies were used for each experiment and data shown is across 3 independent experiments. Data were analyzed by ANOVA and corrected for multiple comparisons using Tukey’s test. Values are mean ± SD and p values across genotypes are shown. (C) Response of flies to varying concentrations of β-ionone, an attractant that binds to Obp28. dMpped^KO flies show no preference in the Y-maze test at low concentrations (0.01mM) of β-ionone, and the behavioral response was restored in dMpped^KO flies with the expression of dMpped restored pan neuronally or specifically in olfactory projection neurons. Flies (w¹¹¹⁸, n = 104; dMpped^KO, n = 89; elav-Gal4>dMpped; dMpped^KO, n = 44; GH>dMPPED;dMPPED^KO, n = 64) were from at least six independent experiments. Data were analyzed by ANOVA and corrected for multiple comparisons using Tukey’s test. Values are mean ± SD and p values are shown. (D) Flies were subjected to desiccation and the time taken for to die was monitored. Restoration of expression of dMpped in dMpped^KO flies restored desiccation resistance to that seen in wildtype flies. Flies per genotype are pooled from at least three independent experiments. Log-rank test used for comparing wild type (w1118, n = 92); dMpped^KO (n = 95) flies; elav-Gal4>dMpped; dMpped^KO (n = 49) and GH146>dMPPED;dMPPED^KO, n = 50. p values across genotypes are shown in the graph.

Fig 5. Mammalian ortholog of dMpped regulates lifespan in flies.

(A) Expression of mMPPED1 and mMPPED2 in mouse tissues. RNA was prepared from the indicated tissues, and expression of mMPPED1, and mMPPED2 monitored following reverse transcription and PCR using specific primers. Expression of β-actin was used to check equivalent cDNA synthesis across tissues. (B) Western blot analysis with purified mMPPED1, mMPPED2 and lysates prepared from total mouse brain or homogenates prepared from the hippocampus and cortex using a monoclonal antibody raised to rat MPPED2. Blots were probed with the MPPED monoclonal antibody and protein loaded in each lane normalized to tubulin. Recombinant, purified, histidine-tagged mMPPED1 and mMPPED2 were used as controls and migrate higher than the endogenous protein present in brain lysates due the hexahistidine tag at the N-terminus of the proteins that facilitated purification. Data is representative of experiments performed with two independently prepared homogenates. The two bands seen in brain homogenates migrate at sizes predicted for mMPPED1 and mMPPED2. (C) Immunocytochemical analysis to show expression of mMPPED in neurons prepared from the cortex of 1-day old mice. (D) Western blot performed homogenates prepared from the brain of flies of the indicated genotypes. Blots were probed either with the monoclonal antibody raised to mammalian Mpped2 or the polyclonal antibody raised to dMpped. Protein loading was normalized by using an antibody to tubulin. The data shown is representative of experiments performed with independently prepared homogenates at least twice. (E) Flies (w¹¹¹⁸, n = 66; dMppedKO, n = 66; elav-Gal4>MPPED2; dMpped^KO, n = 34) were from three independent experiments. Log-rank test was used for comparing across genotypes. p-values across genotypes are shown in the Figure.

Fig 6. Schematic showing pleiotropic roles of dMPPED.

Class III metallophosphoesterases are evolutionarily conserved and members of the MPPED family are neuronally expressed in the brain of mammals and flies. dMPPED^KO flies show misregulation of Obps, elevated levels of cAMP and cGMP and higher expression of AMPs. All these changes can influence longevity, with dMPPED^KO flies showing a decreased life span. The Figure was created with BioRender.com.